Immunomodulating compositions and methods of use thereof

ABSTRACT

The invention is directed to β1-6 glucans, compositions, diagnostic kits, and devices comprising the same, and methods of use thereof in modulating immune response and treating, delaying progression of, reducing the incidence or severity of cancer, infection, inflammation, and autoimmune diseases. The β1-6 glucans of certain embodiments of the invention are enriched for O-acetylated groups and/or conjugated to a solid support or linked to a targeting moiety. The β1-6 glucans of certain embodiments of the invention recruit immunoglobulin G antibodies to mediate complement and neutrophil killing. The conjugated β1-6 glucans of certain embodiments of the invention are targeted to cells to stimulate the immune response at the target location by activating complement-mediated lysis and recruitment of neutrophils.

RELATED APPLICATIONS

This application is a continuation of application Ser. No. 12/990,066,filed Oct. 28, 2010, which is a national phase application under 35 USC§371 of PCT International Application No. PCT/US2009/042117, filed Apr.29, 2009, which claims the benefit of priority to United StatesProvisional Patent Application Ser. No. 61/071,437, filed Apr. 29, 2008;which are hereby incorporated by reference in their entirety.

GOVERNMENT INTEREST STATEMENT

The invention was made in whole or in part with government support underGrant Number GM035010-22 awarded by the National Institute of Health.The government has certain rights in the invention.

BACKGROUND OF THE INVENTION

The cell walls of fungi evoke a powerful immuno-stimulatory response,and have been proposed for use as potential anti-infective andanti-tumor drugs. Fungal cells can also activate dendritic cells andprime class II restricted antigen specific T cell responses. Themajority of the cell wall (50-60%) of pathogenic (Candida albicaus) andnon-pathogenic fungi (Saccharomyces cerevisiae) is composed of an innerlayer of β-glucan β-1,3- and β-1,6-glucan) covalently linked to avariety of cell surface mannoproteins [Klis, F. M. et al. Med Mycol 39Suppl 1, 1-8, 2001; Klis, F. M. et al., FEMS Microbiol Rev 26, 239-56,2002].

Recognition of β-glucans by macrophages is carried out mainly throughDectin-1 with cooperation of TLRs, including TLR2 [Brown, G. D. et al.Nature 413, 36-7, 2001]. Dectin-1 activity is inhibited by β-1,3-glucansand β-1,6-glucans, with the β-1,3-glucan laminarins having the highesteffect. However, oligosaccharide microarray results show that Dectin-1binds specifically to β-1,3-glucans. Neutrophils are professionalkillers, whose role in phagocytosis and killing of bacteria and fungi iswell characterized. Neutropenic individuals are much more susceptible tobacterial and fungal infections, with return to normal counts playing animportant role in resolution of infection. Neutrophils, unlikemacrophages, require serum for optimal phagocytosis and killing. Themain opsonic receptors are the complement receptor CR3 and theimmunoglobulin-binding receptor FcγR. CR3 has a lectin domain [Brown, G.D. et al. Immunity 19, 311-5, 2003] that mediates increased neutrophilmotility towards a mixture of β-1,3-glucan and β-1,6-glucan (PGG-glucan)[Wakshull, E. et al. Immunopharmacology 41, 89-107, 1999].

β-1,6-glucans have been found to provide potent anti-fungal activity,and inter alia, possess adjuvant activity and activate complement.

The complement (C) system of humans and other mammals involves more than20 components that participate in an orderly sequence of reactionsresulting in complement activation. Products derived from the activationof C components include non-self recognition molecules C3b, C4b and C5b,as well as the anaphylatoxins C3a, C4a and C5a that influence a varietyof cellular immune responses (Hugh et al (1982) 15th InternationalLeucocyte Culture Conference, Asilomar, Calif. (Abstract); Fujii et al.(1993) Protein Science 2:1301-1312; Morgan et al. (1982) J. Exp. Med.155:1412-1426; Morgan (1993) Complement Today 1:56-75; Morgan et al.(1983) J. Immunol. 130:1257-1261). Complement activation occursprimarily via the “classical” pathway or the “alternative” pathway. Theclassical pathway is initiated by the binding of the first complementcomponent (C1) to immune complexes through C1q, a subcomponent involvedin binding to antibody. The c1 complex is composed of C1q and twohomologous serine proteases, C1r and C1s (1:2:2 molar ratio). Afterbinding to the immune complexes C1q undergoes a conformational changeresulting in the conversions of C1r and C1s to their activated forms.Activated C1s cleaves C4 and C2 to generate a complex of their fragmentsC4b2a, which in turn cleaves C3 into C3a and C3b. C3b binds to immunecomplexes.

The alternative pathway is activated without involvement of antibody.C3b molecules generated from C3 by interaction of C3 with two serineproteases, factors B and D, are deposited on the microbial surface whereactivation of C3 is amplified. C3b produced by activation of eitherpathway acts as a central molecule in the subsequent formation ofmembrane attack complexes that can lyse microbes and also as an opsonin.

It is unknown whether β-1,6-glucans produce a robust immune response inall subjects and by what mechanism such response is generated.

SUMMARY OF THE INVENTION

This invention provides, in one embodiment, a diagnostic method fordetermining the responsiveness of a subject to a glucan-based vaccine oradjuvant, the method comprising:

-   -   assessing relative immunoglobulin G (IgG) 1, 2, 3 and 4 isotype        titers in a subject exposed to a glucan; and    -   correlating the presence of high IgG1 or IgG2 or IgG3, or a        combination thereof, versus IgG4 with responsiveness to a        glucan-based vaccine or adjuvant.

In one embodiment, the method further comprises contacting a cell in thesubject with a glucan-based vaccine or adjuvant prior to assessingrelative IgG isotype titers, which in one embodiment comprisesO-acetylated glucan, and in another embodiment, comprises glucanisolated or derived from a lichen or a fungus, wherein the fungus isoptionally a yeast, which in one embodiment is isolated or derived fromUmbilicariaceae. In another embodiment, the glucan-based vaccine oradjuvant comprises chemically synthesized or acetylated glucan, and inanother embodiment, the glucan-based vaccine or adjuvant comprisesglucan conjugated to a particle.

In one embodiment, the method further comprises administering a cytokineto said subject, which in one embodiment comprises interleukin 2,interleukin 12, interferon-γ, or a combination thereof

In another embodiment, this invention provides a diagnostic kit fordetermining the responsiveness of a subject to a glucan-based vaccine oradjuvant, said kit comprising:

-   -   a glucan which corresponds to or is homologous to glucan in said        vaccine or adjuvant;    -   reagents for detecting relative immunoglobulin G (IgG) 1, 2, 3        and 4 isotype titers in a subject sample; and    -   optionally a series of standards derived from positive and        negative responders to said glucan-based vaccine or adjuvant.

According to this aspect of the invention, and in one embodiment, theglucan is attached to a substrate, which in one embodiment is amicrotiter plate or in another embodiment is a bead. In one embodiment,the reagents comprise a detectable marker which renders the detectionsemi-quantitative.

In another embodiment this invention provides a method of stimulating animmune response in a subject, comprising administering to the subjectpurified β-1-6-glucan and an agent, which biases antibody production toyield relatively greater amounts of immunoglobulin G (IgG) 1, 2 or 3versus immunoglobulin G (IgG) 4.

According to this aspect of the invention, and in one embodiment, thesubject is administered the purified β-1-6-glucan and the agentconcurrently, or in another embodiment the subject is administeredpurified β-1-6-glucan and the agent sequentially. In another embodimentthe subject is administered said purified β-1-6-glucan and said cytokineeach at least two times.

In one embodiment, the agent is a cytokine, which in one embodiment isinterleukin-2, interleukin-12 or interferon-γ or a combination thereof.In another embodiment, the agent downmodulates interleukin-4 orinterleukin-10 production or interferes with interleukin-4 orinterleukin-10 activity.

In one embodiment, the subject is further exposed to an antigenassociated with a target of the immune response, which in one embodimentis a tumor-associated antigen. According to this aspect of theinvention, and in one embodiment, the subject has a hyperplastic orpreneoplastic lesion, and in another embodiment, the method treats,delays progression of, prolongs remission of, or reduces the incidenceor severity of cancer in the subject. In another embodiment, the subjecthas cancer. In another embodiment, the subject has not been diagnosedwith cancer. In another embodiment, the subject has not been diagnosedwith a tumor.

In another embodiment, the antigen is derived from a pathogen, which inone embodiment is a fungus. In one embodiment, the method treats, delaysprogression of, prolongs latency of, or reduces the incidence orseverity of infection in the subject.

In one embodiment, the invention provides for use of a glucan-basedvaccine or adjuvant, or a glucan, which comprises a particle comprisingβ1-6 glucan. In certain embodiments, the particle consists of at least10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99%β1-6 glucan by dry weight. In certain embodiments, the particle consistsessentially of β 1-6 glucan. Optionally, in certain embodiments, theβ1-6 glucan is enriched for O-acetylated groups. The invention furtherprovides methods and kits as described herein, which may make use of anyof the afore-mentioned particles, or a composition containing any of theafore-mentioned particles.

In one embodiment, the β1-6 glucan is enriched for O-acetylated groups,which in one embodiment contains at least 25% by weight O-acetylatedglucan.

In another embodiment, the glucan-based vaccines or adjuvants, orglucans for use according to this invention comprise β-1,3 glucanshaving β-1,6 glucan branches (also referred to as beta 1,3/1,6, glucanor beta-1,6-branched beta-1,3-glucan) wherein at least some of the β-1,6glucan branches are enriched for O-acetylated groups. In anotherembodiment the invention makes use of a composition comprising (i) β 1-6glucan enriched for O-acetylated groups; and (ii) beta-1,6-branchedbeta-1,3-glucan.

In any embodiment of this invention, with regard to the term“contacting” or grammatical forms thereof, the contacting may occureither outside the body of a subject or within the body. In oneembodiment, cells, which in some embodiments are neutrophils, areremoved from a subject, contacted with the described agents orcomponents or compositions, and then administered to the subject at asubsequent point in time. The cells (which, in some embodiments, areneutrophils or in other embodiments, other immune system cells, such asother professional antigen presenting cells, such as macrophages ordendritic cells, monocytes, NK cells, B cells or others) are contactedoutside the body with an agent/component or composition of thisinvention and are then returned to the subject at which point anotheragent is administered to the subject, or another cell population iscontacted outside the body with an agent/component or composition ofthis invention and are then returned to the subject. The suitable periodof time could be, e.g., after the therapy has been administered and thedesired results are not obtained, or for example, greater effects aredesired, for example, immunoglobulin G4 (IgG4) production is too high,and following repeat administration of a cytokine, and subsequentexposure to the glucans, greater IgG1, 2 or 3 is produced.

In some embodiments, the invention provides kits which comprise asubstrate comprising a glucan. In one embodiment, the substrate is apart of, or in the form of a microparticle, nanoparticle, microtiterplate, or any appropriate material for such diagnostic assays, forexample, suitable for automated assay.

One aspect of the invention relates to a method for detecting the amountof immunoglobulin G (IgG) antibodies secreted in response toβ-1,6-glucan; said method comprising the steps of:

obtaining a first blood sample from a subject prior to challenge withβ-1,6-glucan;

obtaining a second blood sample from a subject after challenge withβ-1,6-glucan;

allowing a sufficient time to generate a signal readable in solution anddetecting the signal in

-   -   solution of IgG antibodies binding in an assay that quantifies        or measures the presence of said IgG antibodies:    -   calculating said signal of IgG antibody binding from said first        and second blood samples to determine the amount of IgG antibody        secretion, wherein the difference between the signals indicate        the amount of IgG antibodies produced in response to        β-1,6-glucan; and comparing said difference to a positive and        negative control; wherein said difference over a threshold        determines the responsiveness of a subject to β-1,6-glucan.

In certain embodiments, said antibodies comprise whole immunoglobulin Gor fragments thereof.

In certain embodiments, said antibodies may comprise isotypes selectedfrom the group consisting of IgG 1, IgG2, IgG3, and IgG4.

In certain embodiments, said antibodies are IgG2.

In certain embodiments, said subject may be human.

In certain embodiments, said β-1,6-glucan is isolated from a lichen,fungus, or yeast.

In certain embodiments, said β-1,6-glucan is isolated fromUmbilicariaceae.

In certain embodiments, said β-1,6-glucan comprises O-acetylatedβ-1,6-glucan.

In certain embodiments, said-1,6-glucan comprises chemicallysynthesized, genetically engineered, or O-acetylated β-1,6-glucan.

In certain embodiments, the amount of IgG antibodies is determine byenzyme-linked immunoassays, radioimmunoassays, immunoprecipitations,fluorescence immunoassays, chemiluminescent assay, immunoblot assays,lateral flow assays, agglutination assays, or particulate-based assays.

In certain embodiments, the pharmaceutical composition further comprisesa β-1,6-glucan linked to a targeting moiety.

Another aspect of the invention relates to a diagnostic kit formeasuring IgG levels, comprising:

-   -   (i) a pharmaceutical composition comprising β-1,6-glucan;    -   (ii) reagents for detecting IgG antibodies bound to said        pharmaceutical composition in a blood sample;    -   (iii) optionally a series of standards derived from positive and        negative responders to said pharmaceutical composition; and    -   (ii) instructions for using the pharmaceutical composition to        detect IgG amounts in a blood sample.

In certain embodiments, said β-1,6-glucan is in solution or lyophilized.

In certain embodiments, said β-1,6-glucan is immobilized on a substrate.

In certain embodiments, said substrate comprises a material selectedfrom the group consisting of plastic, glass, gel, celluloid, paper,magnetic resin, polyvinylidene-fluoride, nylon, nitrocellulose, agarose,latex, and polystyrene.

In certain embodiments, said substrate comprises an ELISA plate,dipstick, microtiter plate, radioimmunoassay plate, beads, agarosebeads, plastic beads, latex beads, immunoblot membranes, and immunoblotpapers.

In certain embodiments, said reagent is conjugated to a detectablemarker.

In certain embodiments, said detectable marker is selected from thegroup consisting of a radioactive label, fluorescent label,chemiluminescent label, chromophoric label, ligand, fluorescein,radioisotope, phosphatase, biotin, biotin-related compound, avidin,avidin-related compound, and peroxidase.

Another aspect of the invention relates to a composition comprising aβ-1,6-glucan linked to a targeting moiety.

In certain embodiments, said glucan is O-acetylated.

In certain embodiments, said glucan contains at least 25% by weightO-acetylated glucan.

In certain embodiments, said glucan is isolated from a lichen, yeast,Fungus, chemically synthesized, or genetically engineered.

In certain embodiments, said glucan is isolated from Umbilicariaceae.

In certain embodiments, said targeting moiety is selected from the groupconsisting of an antibody, antigen, receptor ligand, epitope,polysaccharide, peptide, and any combination thereof.

In certain embodiments, said targeting moiety is an antigen; and theantigen is selected from the group consisting of a glycoprotein,mucoprotein, nucleic acid, carbohydrate, proteoglycan, lipid, mucinmolecule, tumor-associated antigen, and any combination thereof.

In certain embodiments, said antigen is a tumor-associated antigen.

In certain embodiments, said tumor-associated antigen is present on acancer cell selected from the group consisting of an ovarian carcinoma,melanoma, pancreatic carcinoma, colorectal carcinoma, Burkitt'slymphoma, B-cell lymphoma, lung carcinoma, leukemia, breast carcinoma,myeloid carcinoma, colonic adenocarcinoma, gastric carcinoma, embryonalcarcinoma, prostate carcinoma, and endometrial carcinoma.

In certain embodiments, said cancer cell is an ovarian carcinoma; andsaid tumor-associated antigen is a CA125 or CD46.

In certain embodiments, said cancer cell is a melanoma cell; and saidtumor-associated antigen is selected from the group consisting of a p97,gp75, HMW-MAA, ganglioside GD2, ganglioside GD3, MAGE, BAGE, Mart-1R24,ganglioside GM2, and ganglioside GM3.

In certain embodiments, said cancer cell is a pancreatic carcinoma; andsaid tumor-associated antigen present is an ADMR or CRLR.

In certain embodiments, said cancer cell is a carcinoembryoniccarcinoma; and said tumor-associated antigen is a CEA.

In certain embodiments, said cancer cell is a colorectal carcinoma; andsaid tumor-associated antigen is selected from the group consisting of aTAG-72, CO 17-IA, GICA 19-9, CTA-I, LEA, VEP8, VEP9, MyI, VIM-D5,D156-22, C4BP, and DAF.

In certain embodiments, said cancer cell is a Burkitt's lymphoma; andsaid tumor-associated antigen is an antigen-38.13 or CD 19.

n certain embodiments, said cancer cell is a human B-lymphoma; and saidtumor-associated antigen is a CD20 or CD33.

In certain embodiments, said cancer cell is a human lung carcinoma; andsaid tumor-associated antigen is a L6, L20, F3, or CD1 17.

In certain embodiments, said cancer cell is a human leukemia T cell; andsaid tumor-associated antigen is a gp37, neoglycoprotein, sphingolipids, or APO-1.

In certain embodiments, said cancer cell is a breast carcinoma cell; andsaid tumor-associated antigen is an EGFR, HER/neu, CR1, M18, or M39.

In certain embodiments, said cancer cell is a myeloid carcinoma; andsaid tumor-associated antigen is a T5A7 or SSEA-I.

In certain embodiments, said cancer cell is a colonic adenocarcinomacell; and said tumor-associated antigen is a C 14, CO-514, or NS-10.

In certain embodiments, said cancer cell is a gastric carcinoma cell;and said tumor-associated antigen is a mucin, AH6, or FHL-I.

In certain embodiments, said cancer cell is an embryonal carcinoma cell;and said tumor-associated antigen is selected from the group consistingof a Y hapten, Ley, FC 10.2, 4.2, GD3, D1.1, OFA-I, GM2, OFA-2, GD2, andM1:22:25:8.

In certain embodiments, said cancer cell is a prostrate carcinoma; andsaid tumor-associated antigen is a CD55 or MCP.

In certain embodiments, said cancer cell is an endometrial carcinoma;and said tumor-associated antigen is a CD35.

In certain embodiments, said targeting moiety is an epitope; and saidepitope is selected from the group consisting of a T helper cell epitope(TH), chemokine epitope, neutrophilic epitope, MHC class II molecule,and phagocytic epitope.

In certain embodiments, said epitope is a neutrophilic epitope; and saidneutrophilic epitope is selected from the group consisting of aL-selectin, 2-integrins, complement receptor 1 (CR-1),decay-accelerating factor (DAF), C5a receptor, intercellular adhesionmolecule-1 (ICAM-I), and ICAM-3.

In certain embodiments, said epitope is a phagocytic epitope; and saidphagocytic epitope is a Fc receptor.

In certain embodiments, said epitope is a chemokine epitope; and saidchemokine epitope is a CD40, CD80, or CD86.

In certain embodiments, said epitope is a MHC class II molecule; andsaid MHC class II molecule is selected from the group consisting of aCD69, ADAM8, CD14, CD163, CD33, CD63, CD68, CD74, CHIT1, CHSTIO, CSFIR,DPP4, FABP4, FCGRIA, ICAM2, ILIR2, ITGAI, ITGA2. S100A8, and TNFRSF8.

In certain embodiments, said TH epitope is capable of being taken up byan antigen presenting cell (APC) which is capable of being processed bythe APC whereby the APC presents the TH epitope on its surface bound toan MHC class II molecule.

In certain embodiments, said targeting moiety is an antibody; and saidantibody is selected from the group consisting of a monoclonal antibody,polyclonal antibody, bispecific antibody, diabody, tribody, tetrabody,and minibody.

In certain embodiments, said antibody is a monoclonal antibody.

In certain embodiments, said monoclonal antibody is selected from thegroup consisting of an Alemtuzumab (Campath), Bevacizumab (Avastin),Cetuximab (Erbitux), Gemtuzumab (Mylotarg), Ibritumomab (Zevalin),Panitumumab (Vectibix), Rituximab (Rituxan), Tositumomab (Bexxar),Trastuzumab (Herceptin), Palivizumab (Synagis) in A and F protein inrespiratory syncytial virus, and Immunoglobulin G2.

In certain embodiments, said monoclonal antibody is Alemtuzumab(Campath); and said Alemtuzumab (Campath) targets CD52 present onpancreatic carcinoma cells.

In certain embodiments, said monoclonal antibody is Bevacizumab(Avastin); and said Bevacizumab (Avastin) targets VEGF present incolorectal carcinomas.

In certain embodiments, said monoclonal antibody is Cetuximab (Erbitux);and said Cetuximab (Erbitux) targets EGFR present on head neck squamouscell carcinomas or breast carcinomas.

In certain embodiments, said monoclonal antibody is Gemtuzumab(Mylotarg); and said Gemtuzumab (Mylotarg) targets CD33 present onmyeloid leukemias.

In certain embodiments, said monoclonal antibody is Ibritumomab(Zevalin), Panitumumab (Vectibix), Rituximab (Rituxan), or Tositumomab(Bexxar); and said Ibritumomab (Zevalin), Panitumumab (Vectibix),Rituximab (Rituxan), or Tositumomab (Bexxar) targets CD20 present onB-cell lymphomas.

In certain embodiments, said monoclonal antibody is Trastuzumab(Herceptin); and said Trastuzumab (Herceptin) targets HER/neu present onbreast carcinomas.

In certain embodiments, said monoclonal antibody is Palivizumab(Synagis); and said Palivizumab (Synagis) targets A and F proteinpresent on respiratory syncytial viruses.

In certain embodiments, said monoclonal antibody is immunoglobulin G;and said immunoglobin G targets β-1,6-glucan present on fungal orbacterial pathogens.

In certain embodiments, said antibody is a polyclonal antibody; and saidpolyclonal binds to any of the aforementioned antigens or epitopes.

In certain embodiments, said antibody comprises at least oneantigen-binding site that binds to IgG2.

In certain embodiments, said antibody comprises a second, third orfourth antigen-binding site to any of the aforementioned antigens andepitopes.

In certain embodiments, the composition further comprises administeringconcurrently or successively an adjuvant, an antigen, animmunomodulatory compound, or a combination thereof.

Another aspect of the invention relates to a pharmaceutical formulation,comprising a composition of any of the aforementioned compostions ofβ-1,6-glucan linked to a targeting moiety; and a pharmaceuticallyacceptable carrier, diluent, or excipient.

Another aspect of the invention relates to a method for modulating animmune response in a subject, comprising administering to a subject inneed thereof a therapeutically effective amount of a composition of anyof the aforementioned compositions of β-1,6-glucan linked to a targetingmoiety.

In certain embodiments, said immune response to a target cancer cell,infectious agent, pathogen, or site of infection is stimulated.

In certain embodiments, said immune response stimulates or enhances heatshock protein expression.

In certain embodiments, said immune response induces the production ofreactive oxygen species (ROS).

In certain embodiments, said immune response enhances or stimulatesneutrophilic phagocytosis or cytotoxic lysis.

In certain embodiments, said immune response enhances or stimulatescomplement-mediated lysis.

In certain embodiments, the method further comprises administeringconcurrently or successively an adjuvant, an antigen, a peptide, animmuno-stimulatory compound, a therapeutic agent, or a combinationthereof.

In certain embodiments, said therapeutic agent is selected from thegroup consisting of an anti-inflammatory, antiviral, antibiotic,anti-infective, glucan synthesis inhibitors, antiprotozoal,antihistamine, decongestant, antipsychotics, mitotic inhibitor, and anycombination thereof.

In certain embodiments, said therapeutic agent is an antiviral; and saidantiviral is acyclovir, nelfinavir, or virazole.

In certain embodiments, said therapeutic agent is an antibiotic; andsaid antibiotic is selected from a group consisting of ampicillin,penicillin G, penicillins, cephalosporins, aminoglycosidics, macrolides, carbapenem, penem, beta-lactam monocyclic, inhibitors ofbeta-lactamases, tetracyclins, polipeptidic antibiotics,chloramphenicol, fusidic acid, lincomicyn, novobiocine, spectinomycin,poly-etheric ionophores, and quinolones.

In certain embodiments, said therapeutic agent is an anti-infective; andsaid anti-infective is selected from the group consisting ofbenzalkonium chloride, chlorbexidine; dapsone, chloramphenicol,neomycin, cefaclor, cefadroxil, cephalexin, cephradine erythromycin,clindamycin, lincomycin, amoxicillin, ampicillin, bacampicillin,carbenicillin, dicloxacillin, cyclacillin, picloxacillin, hetacillin,methicillin, nafcillin, oxacillin, penicillin, ticarcillin, rifampin,tetracycline, diflunisal, ibuprofen, indomethacin, meclofenamate,mefenamic acid, naproxen, oxyphenbutazone, phenylbutazone, piroxicam,sulindac, tolmetin, aspirin, salicylates, and amphotericin B.

In certain embodiments, said therapeutic agent is a glucan synthesisinhibitor; and said glucan synthesis inhibitor is selected from thegroup consisting of caspofungin, micafungin, anidulafungin (LY303366),econazole, terconazole, fluconazole, voriconazole, and griseofulvin.

In certain embodiments, said therapeutic agent is an antiprotozoal; andsaid antiprotozoal is a metronidazole, tubulazole, thiabendazole, oroxfendazole.

In certain embodiments, said therapeutic agent is an antihistamine; andsaid antihistamine is astemizole, levocabastine, cetirizine, orcinnarizine.

In certain embodiments, said therapeutic agent is a decongestant; andsaid decongestant is pseudoephedrine.

In certain embodiments, said therapeutic agent is an antipsychotic; andsaid antipsychotic is fluspirilene, penfluridole, risperidone, orziprasidone.

In certain embodiments, said therapeutic agent is an mitotic inhibitor;and said mitotic inhibitor is etoposide, colchicine, or vinca alkaloids.

In certain embodiments, said immune response is downregulated orabrogated.

In certain embodiments, said immune response downregulates or abrogatesactivation of interferon, interleukin, tumor necrosis factor,granulocyte-macrophage colony stimulating factor (GM-CSF), macrophagecolony stimulating factor (M-CSF), granulocyte colony stimulating factor(G-CSF), neutrophil activating protein (NAP), macrophage chemoattractantand activating factor (MCAF), RANTES, or macrophage inflammatorypeptides.

In certain embodiments, the method further comprises administeringconcurrently or successively an immunosuppressant.

Another aspect of the invention relates to a method of treating,delaying progression of, prolonging remission of, or reducing theincidence or severity of cancer in a subject, comprising administeringto a subject in need thereof a therapeutically effective amount of acomposition of any of the aforementioned compositions of β-1,6-glucanlinked to a targeting moiety.

In certain embodiments, said immune response to a target cancer cell, ahyperplastic lesion, or a preneoplastic lesion is stimulated.

In certain embodiments, said immune response enhances neutrophilicphagocytosis, stimulates cytotoxic lysis, induces production of ROS,induces the expression of heat shock proteins, or enhancescomplement-mediated lysis.

In certain embodiments, the method further comprises administeringconcurrently or successively a chemotherapeutic agent, a cytotoxicagent, an anti-neoplastic agent, or a combination thereof.

In certain embodiments, said cytotoxic agent is selected from the groupconsisting of an ErbB receptor inhibitor, VEGF receptor inhibitor,tyrosine kinase inhibitor, protein kinase A inhibitor, anti-angiogenicagent, anti-hormonal agent, and cytokine.

In certain embodiments, said chemotherapeutic agent is selected from thegroup consisting of a cisplatin, doxorubicin, gemcitabine, docetaxel,paclitexel, and belomycin.

In certain embodiments, said antineoplastic agent is selected from thegroup consisting of spiroplatin, cisplatin, carboplatin, methotrexate,fluorouracil, adriamycin, mitomycin, ansamitocin, bleomycin, cytosinearabinoside, arabinosyl adenine, mercaptopolylysine, vincristine,busulfan, chlorambucil, melphalan, PAM, L-PAM, phenylalanine mustard,mercaptopurine, mitotane, procarbazine hydrochloride actinomycin D,daunorubicin hydrochloride, doxorubicin hydrochloride, paclitaxel andother taxenes, rapamycin, manumycin A, TNP-470, plicamycin, mithramycin,aminoglutethimide, estramustine phosphate sodium, flutamide, leuprolideacetate, megestrol acetate, tamoxifen citrate, testolactone, trilostane,amsacrine (m-AMSA), asparaginase (L-asparaginase) Erwina asparaginase,interferon .alpha.-2a, interferon .alpha.-2b, teniposide (VM-26),vinblastine sulfate (VLB), vincristine sulfate, bleomycin sulfate,hydroxyurea procarbazine, and dacarbazine.

In certain embodiments, the method further comprises administeringconcurrently or successively an adjuvant, an antigen, animmuno-modulatory compound, or a combination thereof.

Another aspect of the invention relates to a method of treating,delaying progression of, or reducing the incidence or severity of aninfection in a subject, comprising administering to a subject in needthereof a therapeutically effective amount of any of the aforementioncompositions of β-1,6-glucan linked to a targeting moiety.

In certain embodiments, said immune response to a target parasite,virus, fungus, pathogen, bacteria, or infectious agent is stimulated.

In certain embodiments, said immune response enhances neutrophilicphagocytosis, stimulates cytotoxic lysis, induces production of ROS,induces the expression of heat shock proteins, or enhancescomplement-mediated lysis.

In certain embodiments, the method further comprises administeringconcurrently or successively an antibiotic, an antiviral, ananti-infective, an antiprotozoal, an adjuvant, an antigen, animmuno-modulatory compound, or a combination thereof.

Another aspect of the invention relates to a method of treating,delaying progression of, reducing the incidence or severity ofinflammation in a subject, comprising administering to a subject in needthereof a therapeutically effective amount of any of the aforementionedcompositions of β-1,6-glucan linked to a targeting moiety.

In certain embodiments, said inflammation at a target inflammatory fociis downregulated or abrogated.

In certain embodiments, the method further comprises administeringconcurrently or successively an adjuvant, an antigen, a peptide, animmuno-stimulatory compound, an anti-inflammatory agent, or acombination thereof.

In certain embodiments, said anti-inflammatory agent is selected fromthe group consisting of betamethasone, prednisolone, piroxicam, aspirin,flurbiprofen, and(+)-N-{4-[3-(4-fluorophenoxy)phenoxy]-2-cyclopenten-1-yl}-N-hyroxyurea.

Another aspect of the invention relates to a method of treating,delaying progression of, or reducing the incidence or severity of anautoimmune response in a subject, comprising administering to a subjectin need thereof a therapeutically effective amount of any of theaforementioned compositions of β-1,6-glucan linked to a targetingmoiety.

In certain embodiments, said autoimmune response to a targettransplanted tissue, transplanted cells, autoantigen, or HIV infectionis downregulated or abrogated.

In certain embodiments, said autoimmune response downregulatesactivation of interleukin, tumor necrosis factor, or interferon.

In certain embodiments, the method further comprises administeringconcurrently or successively an immunosuppressant.

Another aspect of the invention relates to a composition comprisingpurified β-1,6-glucan linked to a targeting moiety, wherein thecomposition is a pharmaceutical composition, a food or food product, afood supplement, or a cosmetic composition.

In certain embodiments, the composition comprises β-1,6-glucan linked toa targeting moiety that has been processed to increase its ability tomodulate the immune response relative to unprocessed β-1,6-glucan.

In certain embodiments, at least 95% of the glucan contained in thecomposition is β-1,6-glucan linked to a targeting moiety.

Another aspect of the invention relates to a micelle comprisingβ-1,6-glucan linked to a targeting moiety, wherein said β-1,6-glucan isoptionally O-acetylated.

Another aspect of the invention relates to a composition comprisingβ-1,6-glucan linked to a targeting moiety and a biodegradable polymer,wherein said biodegradable polymer degrades to form biologically activesalicylate or alpha-hydroxy acid moieties and said β-1,6-glucan isoptionally O-acetylated.

Another aspect of the invention relates to a particle comprising amicrosphere linked to any of the aforementioned compositions ofβ-1,6-glucan linked to a targeting moiety and a biodegradable polymer.

Another aspect of the invention relates to a medical device comprisingthe composition of any of the aforementioned compositions ofβ-1,6-glucan linked to a targeting moiety and a biodegradable polymer.

In certain embodiments, at least a portion of a surface is coated with acomposition comprising a β-1,6-glucan linked to a targeting moiety.

In certain embodiments, the device is selected from the group consistingof a catheter, stent, valve, pacemaker, central line, pessary, tube,shunt, feeding tube, drain, and orthopedic hardware devices.

In certain embodiments, the composition comprises a coating layercomprising a polymer and β-1,6-glucan linked to a targeting moiety.

In certain embodiments, the composition comprises a coating layercomprising a polymer and β-1,6-glucan linked to a targeting moiety,wherein the polymer is biodegradable.

Another aspect of the invention relates to a method of treating asubject comprising implanting or introducing any of the aforementionedmedical devices into the body of a subject in need thereof.

Another aspect of the invention relates to a coated material comprising:(a) a substrate; and (b) a composition comprising a β-1,6-glucan linkedto a targeting moiety that is physically associated with at least aportion of a surface of said substrate, wherein said composition isoptionally in the form of a gel or film.

In certain embodiments, the composition is a polymer.

In certain embodiments, the composition comprises a biodegradablepolymer.

In certain embodiments, the substrate is composed at least in part ofmetal, ceramic, or polymer.

Another aspect of the invention relates to a method of treating asubject comprising contacting the body of a subject in need thereof withthe coated material of any of the aforementioned coated materials.

All publications, patents, and patent applications mentioned herein arehereby incorporated by reference in their entirety as if each individualpublication or patent was specifically and individually indicated to beincorporated by reference. In case of a conflict between thespecification and an incorporated reference, the specification shallcontrol. Where number ranges are given in this document, endpoints areincluded within the range.

Furthermore, it is to be understood that unless otherwise indicated orotherwise evident from the context and understanding of one of ordinaryskill in the art, values that are expressed as ranges can assume anyspecific value or subrange within the stated ranges, optionallyincluding or excluding either or both endpoints, in differentembodiments of the invention, to the tenth of the unit of the lowerlimit of the range, unless the context clearly dictates otherwise. Wherea percentage is recited in reference to a value that intrinsically hasunits that are whole numbers, any resulting fraction may be rounded tothe nearest whole number.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1E demonstrate engulfment of β-1,6-glucan-coated beads (FIG. 1A(serum) and 1B (IgG-depleted serum)) and ROS production (FIG. 1C) areantibody dependent, irrespective of the presence of complement (FIGS. 1D(Number of SRBC) and 1E (O.D. 414)).

FIGS. 2A-2C demonstrate that β-1,6-glucan antibodies are prevalent innormal adult sera. FIG. 2A is a representative serum of the highresponders, and FIG. 2B shows less efficient engulfment and ROSproduction. FIG. 2C provides data for a medium responder and a highresponder.

FIG. 3A (IgG), FIG. 3B (IgG1), FIG. 3C (IgG2), FIG. 3D (IgG3), and FIG.3E (IgG4) demonstrate that select IgG isotypes influence responsivenessto β-glucan (see legend in FIG. 3F).

FIGS. 4A-4F demonstrate that a β-1,6-glucan-Herceptin conjugate isfunctional. Conjugation of β-1,6-glucan to Herceptin did not affect itsbinding to breast cancer cells (FIG. 4A). Furthermore, the conjugatemediated high C3 deposition (FIG. 4B), suggesting that β-1,6-glucanremained functional. Non-specific isotype control antibodies conjugatedto β-1,6-glucan did not bind breast cancer cells (FIG. 4C), andtherefore, did not mediate complement activation and C3 deposition onthese cells (FIG. 4D). Mixing β-1,6-glucan with the mAb without chemicalconjugation did not mediate C3 deposition on these breast cancer cells(FIG. 4E). C3 deposition was detected on breast cancer cells treatedwith Herceptin conjugated to β-1,6-glucan but not β-1,3-glucan (FIG.4F).

FIGS. 5A-5B demonstrate that a β-1,6-glucan-Herceptin conjugate mediatesthe killing of cancer cells by complement and neutrophils. TheHerceptin-β-1,6-glucan conjugate showed a dose-dependent cytotoxiceffect on the breast cancer cells, whereas the unconjugated Herceptinlacked any effect (FIG. 5A). Furthermore, the Herceptin-β-1,6-glucanconjugate showed an increased neutrophil killing of the cancer cells(FIG. 5B).

It will be appreciated that for simplicity and clarity of illustration,elements shown in the figures have not necessarily been drawn to scale.For example, the dimensions of some of the elements may be exaggeratedrelative to other elements for clarity. Further, where consideredappropriate, reference numerals may be repeated among the figures toindicate corresponding or analogous elements.

DETAILED DESCRIPTION OF THE INVENTION

In the following detailed description, numerous specific details are setforth in order to provide a thorough understanding of the invention.However, it will be understood by those skilled in the art that thepresent invention may be practiced without these specific details. Inother instances, well-known methods, procedures, and components have notbeen described in detail so as not to obscure the present invention.

Glucans are polysaccharides found so far in all studied species oflichenized fungi. Partially O-acetylated pustulans are typical ofUmbilicariaceae, and have been described for several species ofUmbilicaria, such as U. pustulata and U. hirsute, U. angulata, U.caroliniana, and U. polyphylla.

Responsiveness to β-1,6-glucans, were found by the inventors to beantibody-dependent, and robustness of this response was found to beassociated with subjects having particular immunoglobulin G (IgG)isotype expression. Such expression may be useful therefore inpredicting a subject's responsiveness to glucan-based vaccines oradjuvants, and such activity may be useful in modulating immuneresponses.

In one embodiment, the invention provides a diagnostic method fordetermining the responsiveness of a subject to a glucan-based vaccine oradjuvant, the method comprising:

-   -   assessing relative immunoglobulin G (IgG) 1, 2, 3 and 4 isotype        titers in a subject exposed to a glucan; and    -   correlating the presence of high IgG1 or IgG2 or IgG3, or a        combination thereof, versus IgG4 with responsiveness to a        glucan-based vaccine or adjuvant.

In some embodiments, such method may be practiced on any biologicalfluid of or sample isolated from the subject. In some embodiments, suchmethod is practiced on serum or plasma isolated from a subject

It is to be appreciated that there are many methods known in the art forassessing IgG isotype titers, for example, a modified ELISA assay may beconducted, where a glucan or a fragment thereof is adsorbed to asubstrate, which is then incubated with the fluid or sample from thesubject, such that glucan-induced antibody isotype may be determined byprobing with enzyme-labeled anti-IgG subclass-specific secondaryantibody. Other assays, such as the Ouchterlony (double diffusion)assay, or other single-step identification methods, which arecommercially available, may be utilized, as well.

In one embodiment, the method specifically assesses relative differencesin IgG isotype expression in a given sample, such that the relativeincrease in IgG1, IgG2 or IgG3 versus that of IgG4 serves as anindicator for responsiveness to the glucan-based vaccine or adjuvant.

In one embodiment, the method further comprises contacting a cell in thesubject with a glucan-based vaccine or adjuvant prior to assessingrelative IgG isotype titers, which in one embodiment comprisesO-acetylated glucan, and in another embodiment, comprises glucanisolated or derived from a lichen or a fungus, wherein the fungus isoptionally a yeast, which in one embodiment is isolated or derived fromUmbilicariaceae.

In some embodiments, the methods of this invention make use of acomposition comprising purified β 1-6 glucan, wherein the compositionis, in various embodiments of the invention, a pharmaceuticalcomposition, a food or food product, a food supplement, or a cosmeticcomposition. The composition is, in some embodiments, distinct fromcompositions such as pustulan or preparations of fungal cell walls. Incertain embodiments of the invention at least 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, 95%, 98%, or 99% of the glucan contained in thecomposition by weight is β1-6 glucan. In certain embodiments between 20%and 50% of the glucan contained in the composition is β 1-6 glucan. Incertain embodiments between 50% and 100% of the glucan contained in thecomposition is β 1-6 glucan. In one embodiment of any of thecompositions or methods of the invention, the glucan contains from about15% to about 30% by weight β 1-6 glucan. In another embodiment of any ofthe compositions or methods of the invention, the glucan contains fromabout 10% to about 35% by weight β 1-6 glucan, or in another embodiment,from about 20% to about 50% by weight β 1-6 glucan, or in anotherembodiment, from about 25% to about 60% by weight β 1-6 glucan, or inanother embodiment, from about 35% to about 80% by weight β 1-6 glucan,or in another embodiment, from about 18% to about 35% by weight β 1-6glucan, or in another embodiment, from about 15% to about 75% by weightβ 1-6 glucan. In certain embodiments, said glucan is a mixture ofoligomers or polymers, wherein the β-1,6-glucan is greater than about10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% by weightof those oligomers or polymers. In certain embodiments of the invention“weight” refers to “dry weight”. In other embodiments “weight” refers tototal weight. In certain embodiments of the invention the β1-6 glucan isprocessed. Such processing may comprise, for example, deacetylation,treatment with enzymes that digest glucans other than β 1-6 glucan,limited digestion with enzymes that digest β 1-6 glucan, selection ofparticular molecular weight ranges, etc. In certain embodiments,processing comprises separation from other glucans, e.g., α-glucans, β1-3 glucans, etc. In certain embodiments the processing comprisesremoving β 1-6 glucan side chains from β 1-3 glucans and optionallyseparating the β 1-6 glucans side chains. In certain embodiments thecomposition comprises processed β1-6 glucan, wherein the processed β 1-6glucan exhibits enhanced ability to desirably modulate the immuneresponse relative to unprocessed glucan or relative to unprocessed β 1-6glucan.

This invention makes use, in one embodiment, of β1-6 glucan enriched forO-acetylated groups. In one embodiment, in any of the preparations foruse according to the methods of the invention, the glucan contains atleast 25% by weight O-acetylated glucan. In one embodiment, in any ofthe preparations for use according to the methods of the invention, theglucan contains from about 15% to about 30% by weight O-acetylatedglucan. In another embodiment, in any of the preparations for useaccording to the methods of the invention, the glucan contains fromabout 10% to about 35% by weight O-acetylated glucan, or in anotherembodiment, from about 20% to about 50% by weight O-acetylated glucan,or in another embodiment, from about 25% to about 60% by weightO-acetylated glucan, or in another embodiment, from about 35% to about80% by weight O-acetylated glucan, or in another embodiment, from about18% to about 35% by weight O-acetylated glucan, or in anotherembodiment, from about 15% to about 75% by weight O-acetylated glucan.In other embodiments, the glucan contains between about 75% and 100% byweight O-acetylated glucan, e.g., between 75% and 90%, or between 90%and 100% by weight O-acetylated glucan. In one embodiment, in any of thepreparations for use according to the methods of the invention, theglucan contains approximately that percentage of O-acetylated glucoseunits (by weight or number, in various embodiments of the invention)that would result from digestion of a naturally occurring β1-6 glucan(e.g., pustulan or any other β 1-6 glucan mentioned herein) with a β1-6endoglucanase for a time sufficient to digest at least 90% by weight ofthe β1-6 glucan to oligosaccharides comprising 5 or fewer glucose unitsfollowed by (i) removal of those oligosaccharides comprising 5 or fewerglucose residues from the composition or (ii) isolation of a portion ofthe resulting composition having a molecular weight greater than 5 kD,or in some embodiment greater than 10, 20, 30, 50, or 100 kD.

In some embodiments, the term “enriched for O-acetylated residues”refers to the enhanced % of O-acetylated sites in individual glucoseunits within the glucan molecule, enhanced % of O-acetylated glucoseunits within the glucan molecule, or a combination thereof, as comparedto a native glucan molecule. In one embodiment, reference to glucanpreparations enriched by a particular weight percent for O-acetylatedglucan, refers to preparations comprising an enhanced % of O-acetylatedsites in individual glucose units within the glucan molecule, anenhanced % of O-acetylated glucose units within the glucan molecule, ora combination thereof, as compared to a glucan molecule.

Glucans derived from different sources may comprise varying amounts ofO-acetylation in terms of O-acetylated sites in individual glucoseunits, O-acetylated glucose units within the glucan molecule, or acombination thereof. According to this aspect of the invention, the term“enriched for O-acetylated glucan” refers, in some embodiments, toenhanced O-acetylation as described herein, between the reference sourcefrom which the glucan is derived, and may not represent an overallenrichment as compared to any glucan source.

In one embodiment, the term “enriched for O-acetylated glucan” refers,to an enrichment of at least 25% by weight of the glucan chains, whichare O-acetylated on at least one glucose unit, or at least 25% of theglucose units present in the glucan in the composition are O-acetylated,or a combination thereof. In some embodiments, at least 25% of theglucose units in at least 1%, or in another embodiment, at least 5% ofthe beta glucan chains are O-acetylated. In other embodiments between25% and 35%, between 25% and 50%, between 25% and 75%, between 15% and45%, between 20% and 60%, between 35% and 80%, or others of the glucoseunits in at least 5% of the beta glucan chains are O-acetylated, etc. Inother embodiments, embodiments between 25% and 35%, between 25% and 50%,between 25% and 75%, between 15% and 45%, between 20% and 60%, between35% and 80%, or others of the glucose units, in at least 10% of the betaglucan chains, or in another embodiment, in at least 15% of the betaglucan chains, or in another embodiment, in at least 20% of the betaglucan chains, are O-acetylated.

In one embodiment, the glucan is isolated or derived from a lichen,which in one embodiment is from the genus Umbilicariaceae. In oneembodiment, the glucan is isolated from a fungus. In one embodiment, theglucan is isolated from yeast, or in another embodiment the glucan ischemically synthesized or acetylated. In another embodiment, the glucanis conjugated to a solid support.

Glucans are glucose-containing polysaccharides found inter alia infungal cell walls, α-glucans include one or more α-linkages betweenglucose subunits and β-glucans include one or more α-linkages betweenglucose subunits

β1,6-glucans occur frequently in fungi but are rarer outside fungi. Theglucan used in accordance with the invention comprises β 1,6 glucan. Insome embodiments, the f-glucans are derived from Umbilicariaceae, suchas U. pustulata and U. hirsute, U. angulata, U. caroliniana, and U.polyphylla.

In some embodiments, the β-glucans are derived from Candida, such as C.albicans. Other organisms from which β-glucans may be used includeCoccidioides immitis, Trichophyton verrucosum, Blaslomyces dermatidis,Cryptococcus neofirmans, Histoplasma capsulatlum, Saccharomycescerevisiae, Paracoccidioides brasiliensis, and Pvthiumn insidiosum. Insome embodiments, the β-glucans are chemically or enzymaticallysynthesized, as is known in the art, or in other embodiments, theβ-glucans are derived from any species producing the same, andchemically or enzymatically altered, for example, to increaseO-acetylation of the molecule.

In some embodiments, the β-glucans are fungal glucans. A ‘fungal’ glucanwill generally be obtained from a fungus but, where a particular glucanstructure is found in both fungi and non-fungi (e.g., in bacteria, lowerplants or algae) then the non-fungal organism may be used as analternative source.

Full-length native β-glucans are insoluble and have a molecular weightin the megadalton range. In some embodiments, this invention providessoluble β-1,6-glucan. In some embodiments, this invention providessoluble O-acetylated β-1,6-glucan. Solubilization may be achieved byfragmenting long insoluble glucans, in some embodiments. This may beachieved by, for example, hydrolysis or, in some embodiments, bydigestion with a glucanase (e.g., with a β-1,3 glucanase or limiteddigestion with a β-1,3 glucanase). In other embodiments, glucans can beprepared synthetically, for example, and in some embodiments, by joiningmonosaccharide building blocks. O-acetylation of such glucans canreadily be accomplished by methods known in the art. Such methods mayinclude chemical and/or enzymatic acetylation, such as are known in theart.

There are various sources of fungal β-glucans. For instance, pureβ-glucans are commercially available e.g., pustulan (Calbiochem) is aβ-1,6-glucan purified from Umbilicaria papullosa. β-glucans can bepurified from fungal cell walls in various ways, for example, asdescribed in Tokunaka et al. [(1999) Carbohydr Res 316:161-172], and theproduct may be enriched for β-1,6-glucan moieties, or O-acetylatedβ-1,6-glucan moieties, by methods as are known in the art.

One of ordinary skill in the art will be able to identify or selectappropriate methods to enrich for β-1,6-glucan moieties and/or forO-acetylated β-1,6-glucan. In one embodiment, O-acetylation ofbeta-glucan is performed chemically. For example, polysaccharides (50mg) are dried in a speed vac centrifuge and resuspended in 1.5 mL ofacetic anhydride (Mallinderockdt). After resuspension, a few crystals of4-dimethylaminopyridine (Avocado Research Chemist, Ltd) are added ascatalyst. The reaction is allowed to proceed at room temperature for 5,20, or 120 minutes and then stopped with 2 volumes of water. Afterwardsthe samples are dialyzed overnight against water. It will be appreciatedthat this process could be varied or scaled up, as evident to one ofskill in the art. In other embodiments, methods for separatingO-acetylated β-1,6-glucan include one or more of the following steps,which could be performed in various orders: (a) separation based onhigher hydrophobicity, such as binding to any hydrophobic matrix/resin;(b) separation based on digestion with a suitable endo- or exo-glucanaseor combination thereof, wherein the O-acetylated β-1,6-glucan isresistant to digestion; (c) affinity separation using antibodies orother moieties that bind to β-1,6-glucan or to O-acetyl groups thereon;(d) separation based on molecular weight. In one embodiment,β-1,6-glucan is digested with an enzyme that digests unacetylated and/orlightly acetylated β-1,6-glucan. The resulting material is separatedbased on size or molecular weight and a portion comprising heavilyacetylated glucan is isolated. In some embodiments, β-1,6-glucanpreparations are obtained, digested and O-acetylated oligosaccharidesare separated or in another embodiment, isolated, and used in thepreparation of new compositions. Such compositions represent embodimentsof the β-1,6-glucan preparations enriched for O-acetylated residues ofthis invention.

It is to be understood that the products of any process for preparingenriched O-acetylated β-1,6-glucan preparations are to be considered asappropriate for use in the methods and kits of this invention.

In some embodiments, the glucans for use in the kits and/or according tothe methods of this invention may comprise structural modifications, notpresent in native glucan preparations. Such modifications may comprise,O-acetylation, as described herein. In other embodiments, suchmodifications may comprise methylation, alkylation, alkoylation,sulfation, phosphorylation, lipid conjugation or other modifications, asare known to one skilled in the art. In some embodiments themodification comprises modification (e.g., esterification) with an acidsuch as formic, succinic, citric acid, or other acid known in the art.

In some embodiments, lipid conjugation to any or all free hydroxylgroups may be accomplished by any number of means known in the art, forexample, as described in Drouillat B, et al, Pharm Sci. 1998 January;87(1):25-30, B. N. A. Mbadugha, et al, Org. Lett., 5 (22), 4041-4044,2003.

In some embodiments, methylation may be accomplished and verified by anynumber of means known in the art, for example, as described in Mischnicket al. 1994 Carbohydr. Res., 264, 293-304; Bowie et al. 1984, Carbohydr.Res., 125, 301-307; Sherman and Gray 1992, Carbohydr. Res., 231,221-235; Stankowski and Zeller 1992, Carbohydr. Res., 234, 337-341;Harris, P. J., et al. (1984) Carbohydr. Res. 127, 59-73; Carpita, N. C.& Shea, E. M. (1989) Linkage structure of carbohydrates by gaschromatography-mass spectrometry (GC-MS) of partially methylated alditolacetates. In Analysis of Carbohydrates by GLC and MS (Biermann, C J. &McGinnis, G. D., eds), pp. 157-216. CRC Press, Boca Raton, Fla.

In some embodiments, methylation can be confirmed by GLC offurther-derived TMS ethers, acetates or other esters, coupled MS, ordigestion to monosaccharides, de-O-methylation and analysis byderivatization and GLC/MS, for example as described in Pazur 1986,Carbohydrate Analysis—A Practical Approach, IRL Press, Oxford, pp.55-96; Montreuil et al. 1986, Glycoproteins. In M. F. Chaplin and J. F.Kennedy, (eds.), Carbohydrate Analysis—a Practical Approach, IRL Press,Oxford, pp. 143-204; Sellers et al. 1990, Carbohydr. Res., 207, C1-C5;O'Neill et al. 1990, Pectic polysaccharides of primary cell walls. InP.M. Dey (ed.), Methods in Plant Biochemistry, Volume 2, Carbohydrates,Academic Press, London, pp. 415-441; Stephen et al. 1990, Methods inPlant Biochemistry, Volume 2, Carbohydrates, Academic Press, London, pp.483-522; or Churms 1991, CRC Handbook of Chromatography. Carbohydrates,Volume II, CRC Press, Boca Raton, Fla., USA).

In some embodiments, phosphorylation, optionally including theintroduction of other modifications, and verification of the obtainedproduct may be accomplished by means well known to those skilled in theart, see for example, Brown, D. H., Biochem. Biophys. Acta, 7, 487(1951); Roseman, S., and Daffner. L. Anal. Chem., 28, 1743 (1956);Romberg, A., and Horecker,. B. L., in Methods in enzymology, Vol. I,Academic Press, New York, 1955, p. 323; U.S. Pat. No. 4,818,752.

In some embodiments, glucan sulfation and verification of the obtainedproduct may be accomplished by any of the means well known in the art,for example, as described in Alban, S., and Franz, G. (2001),Biomacromolecules 2, 354-361; Alban, et al. (1992) Arzneimittelforschung42, 1005-1008; or Alban, S., et al. (2001). Carbohydr. Polym. 47,267-276.

Also provided by the invention is use of a micelle comprisingβ-1,6-glucan. In certain embodiments the micelle comprises a complexcomposed of surfactant molecules comprising β-1,6-glucan, which may bedispersed in a liquid colloid. In certain embodiments the surfactantmolecules are amphilic, i.e., they contain both hydrophobic groups(their “tails”) and hydrophilic groups (their “heads”). In certainembodiments the hydrophilic component comprises β-1,6-glucan, optionallymodified according to any one or more ways described herein. In certainembodiments a micelle in aqueous solution forms an aggregate with thehydrophilic “head” regions in contact with surrounding solvent,sequestering the hydrophobic tail regions in the micelle center. Themicelle may be globular and roughly spherical in shape, but in certainembodiments the micelle is an ellipsoid, cylinder, or bilayer. In someembodiments the micelle is a polymeric micelle such as those describedin U.S. Pub. No. 20020035217. In some embodiments the micelleencapsulates an active agent, e.g., a hydrophobic molecule. Exemplaryactive agents include anti-infective agents such as antibacterial,anti-viral, anti-fimungal, anti-parasite agents; chemotherapeutic agentsfor treatment of cancer; a cytokine, antigen, etc.

The invention further provides β-1,6-glucan that is modified byconjugating a lipid thereto, wherein the modification in someembodiments allows for creation of a micelle comprising β-1,6-glucanhaving the lipid attached thereto. The lipid may be a straight chain orbranched, optionally substituted, hydrocarbon. In some embodiments thelipid comprises a fatty acid. In some embodiments the lipid, e.g., fattyacid, contains between 4 and 26 or between 4 and 40 carbon atoms.

Also provided by the present invention is use of or a kit including aparticle comprising β-1,6-glucan covalently or noncovalently linked to aparticle comprising or consisting essentially of yeast glucan. Alsoprovided is β-1,6-glucan comprising a reactive moiety able to react witha functional group of a yeast glucan to form a covalent bond. The yeastglucan may comprise β-1,6-glucan, β-1,3-glucan, other glucans, or acombination thereof.

In another embodiment, this invention provides a diagnostic kit fordetermining a subject's responsiveness to a glucan-based vaccine oradjuvant, said kit comprising:

-   -   a glucan which corresponds to or is homologous to glucan in said        vaccine or adjuvant;    -   reagents for detecting relative immunoglobulin G (IgG) 1, 2, 3        and 4 isotype titers in a subject sample; and    -   optionally a series of standards derived from positive and        negative responders to said glucan-based vaccine or adjuvant.

According to this aspect of the invention, and in one embodiment, theglucan is attached to a substrate, which in one embodiment is amicrotiter plate or in another embodiment is a bead. In one embodiment,the reagents comprise a detectable marker which renders the detectionsemi-quantitative.

In some embodiments, the subject has been exposed to environmentalglucans, and the diagnostic is for determining responsiveness of thesubject to a particular glucan-based vaccine. According to this aspect,the kit will comprise a glucan which corresponds to or is a fragment of,or is highly homologous to the glucan in the vaccine for whichresponsiveness is being determined.

According to this aspect, and in one embodiment, the term “homology”,when in reference to a glucan as herein described, indicates apercentage of structural identity or identity in terms of composition orcontent in the candidate molecule as compared to a corresponding glucanreference molecule.

In one embodiment, the terms “homology”, “homologue” or “homologous”, inany instance, indicate that the molecule referred to, exhibits at least70% correspondence with the reference molecule. In another embodiment,the glucan molecule exhibits at least 72% correspondence with thereference molecule. In another embodiment, the glucan molecule exhibitsat least 75% correspondence with the reference molecule. In anotherembodiment, the glucan molecule exhibits at least 77% correspondencewith the reference molecule. In another embodiment, the glucan moleculeexhibits at least 80% correspondence with the reference molecule. Inanother embodiment, the glucan molecule exhibits at least 82%correspondence with the reference molecule. In another embodiment, theglucan molecule exhibits at least 85% correspondence with the referencemolecule. In another embodiment, the glucan molecule exhibits at least87% correspondence with the indicated sequence. In another embodiment,the glucan molecule exhibits at least 90% correspondence with thereference molecule. In another embodiment, the glucan molecule exhibitsat least 92% correspondence with the reference molecule. In anotherembodiment, the glucan molecule exhibits at least 95% or morecorrespondence with the reference molecule. In another embodiment, theglucan molecule exhibits at least 95%-100% correspondence to thereference molecule.

With regard to correspondence to a reference molecule, suchcorrespondence may refer to structural identity or compositionalidentity, in terms of chemical content. In some embodiments, similarlyprepared glucans are utilized in kits of this invention, which arecomparable to those utilized in a glucan-based adjuvant or vaccine,however the glucan utilized in the kit may not have been subjected toall processing steps, which comprise the preparation process for aglucan-based vaccine or adjuvant.

Homology may be determined, in one embodiment, by methods well describedin the art, including immunoblot analysis, or via computer algorithmanalysis of, utilizing any of a number of software packages available,via established methods.

Also provided by the invention is a kit comprising or use of acomposition comprising β-1,6-glucan and a biodegradable polymer. In someembodiments the biodegradable polymer comprises biologically activesubunits. The term “biodegradable” refers, in some embodiments, to amaterial, which is degraded, i.e., broken down into smaller fragments,in the biological environment of the cell or subject in which it isfound. In one embodiment, biodegradation involves the degradation of apolymer into its component subunits, via, for example, enzymatic ornon-enzymatic hydrolysis, digestion, etc. In one embodiment,biodegradation may involve cleavage of bonds (whether covalent orotherwise) in the polymer backbone. In another embodiment,biodegradation may involve cleavage of a bond (whether covalent orotherwise) internal to a side-chain or one that connects a side chain tothe polymer backbone. In some embodiments the degradation products aremetabolizable by the subject. In some embodiments the degradationproducts are usable by the subject for synthesis of larger biomolecules.In some embodiments the degradation products are excreted or otherwiseeliminated by the subject. In some embodiments the polymer and/or itsdegradation products are biocompatible in that they are substantiallynontoxic and do not produce an unacceptable inflammatory or immuneresponse when administered or otherwise introduced into the body of asubject in amounts consistent with the present invention.

The term “biologically active agent” includes, in some embodiments,therapeutic agents that provide a therapeutically desirable effect whenadministered to an animal (e.g., a mammal, such as a human) in effectiveamounts, it being understood that not all subjects will benefit from theagent. In some embodiments the polymer is a polyanhydride, whichoptionally comprises biologically active salicylates and alpha-hydroxyacids. Degradation of the polymer releases said biologically activesalicylates and/or alpha-hydroxy acids. In some embodiments theβ-1,6-glucan is covalently or noncovalently attached to thebiodegradable polymer. Suitable polymers and methods for manufacturethereof are described, e.g., in U.S. Publication No. 20030035787 and20050053577. In certain embodiments the polymer comprises between 10 and1000, or between 50 and 500, or about 100 monomers. In one embodimentthe polymer is Polyaspirin®. Methods of forming a compound in which aβ-1,6-glucan is covalently linked to the polymer will be evident to oneof skill in the art. The β-1,6-glucan could be covalently attached to amonomer prior to polymerization or could be conjugated to a functionalgroup of the polymer following polymerization. In some embodiments theβ-1,6-glucan is covalently attached via a linking group. Exemplarylinking groups are described in U.S. Pub. No. 20050053577, and otherswill be evident to one of skill in the art.

In some embodiments the kits comprise and methods make use of a particlecomprising β-1,6-glucan and the biodegradable polymer. In someembodiments the particle is coated with or impregnated withβ-1,6-glucan. In some embodiments the β-1,6-glucan is covalentlyattached to the polymer. In some embodiments the composition coats animplant or other medical or surgical device as described elsewhereherein.

Further provided are methods of administering a β-1,6-glucan and abiologically active salicylate or alpha-hydroxy acid to a subject, themethod comprising administering a composition comprising β-1,6-glucanand a biodegradable polymer comprising said biologically activesalicylates and/or alpha-hydroxy acids to the subject or implanting orintroducing a device comprising said polymer and said biologicallyactive salicylates and/or alpha-hydroxy acids into a subject.

In some embodiments, this invention provides kits comprising and methodsmaking use of low molecular weight glucans, having a molecular weight ofless than 100 kDa (e.g., less than 80, 70, 60, 50, 40, 30, 25, 20, or 15kDa). In some embodiments, this invention provides oligosaccharidese.g., containing 85 or fewer (e.g., 85, 84, 83, 82, 81, 80, 79, 78, 77,76, 75, 74, 73, 72, 71, 70, 69, 68, 67, 66, 65, 64, 63, 62, 61, 60, 59,58, 57, 56, 55, 54, 53, 52, 51, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41,40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23,22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4)glucose monosaccharide units.

In some embodiments the β-β-1,6-glucan used in the kits or methods ofthis invention comprises or consists essentially of a low molecularweight glucan. In some embodiments of any method of the invention inwhich β-1,6-glucan is utilized, the β-1,6-glucan comprises or consistsessentially of a low molecular weight glucan. Optionally at least someof the low molecular weight β-1,6-glucan in any embodiment of theinvention is enriched for O-acetylated groups.

A common technique in determining linkage type and structure in glucansis carbon-13 nuclear magnetic resonance spectroscopy (13 C-NMR). Thenumber and relative intensities of ¹³C signals in a given spectrum canbe used to determine linkage configurations and positions in glucanpolymers. For example, the chemical shifts (signals) of carbon atomsengaged in the glycosidic bond are shifted strongly downfield (up to 9ppm) compared to the corresponding unlinked carbons.

This invention provides, in some embodiments, kits and uses of acomposition comprising β 1-6 glucan, wherein the glucan is conjugated toa solid support. In one embodiment, the solid support is a bead orparticle.

In one embodiment, the beads or particles or substrates to which glucansare conjugated comprise denatured proteins (e.g., human serum albumin(Benacerraf et al, 1957 Brit. J. Exp. Path, 38:35)), insoluble materials(e.g., carbon black, silica, silicon dioxide, polystyrene, latex), metaloxides (e.g., titanium oxides, iron oxides), and India ink (i.e.,suspension of colloidal carbon particles) (described in Reichard andFilkins, 1984, The Reticuloendothelial System; A Comprehensive Treatise,pp. 73-101 (Plenum Press), and references therein), hydrogels, (forexample as described in US Patent Publication No. 20050191361),sepharose or agarose beads or microparticles. In some embodiments thebeads or microparticles are formed from materials that are biodegradableand non-toxic (e.g., a poly(α-hydroxy acid) such aspoly(lactide-co-glycolide), a polyhydroxybutyric acid, a polyorthoester,a polyanhydride, a polycaprolactone, etc.). The beads or particles ofthe present invention may comprise red blood cells (RBCs) that have beenpurged of their cytoplasm, known as ‘Ghost’ RBCs, bacteria (as bacteriaare cleared by the RES; see, e.g., Benacerraf and Miescher, 1960, Ann NYAcad Sci, 88:184-195), cell fragments, liposomes, bacteriophages,bacteriophage fragments, and viral capsids devoid of the viral nucleicacids (e.g., hepatitis B virus surface antigen particles), etc.

In one embodiment, conjugation to the solid support is via chemicallycross-linking the solid to the glucans of this invention. The chemistryof cross-linking is well known in the art. The nature of thecrosslinking reagent used to conjugate the glucan and the solid (e.g.,bead or particle) can be any suitable reagent known in the art. It is tobe understood that any suitable crosslinking agent may be used with caretaken that the activity of the glucan is preserved.

The particle may be a fragment of a bacteriophage or bacteria.

In certain embodiments the size of the particle is appropriate foringestion by macrophages, neutrophils, or both. The particle can haveany of the compositions described herein. In certain embodiments theinvention provides a population of particles, wherein at least 50% ofthe particles have a size appropriate for ingestion by macrophages,neutrophils, or both.

The invention provides populations of particles, wherein at least 50%,75%, or 90% of the particles fall within a desired size range. Incertain embodiments the desired size ranges within ±10%, ±20%, ±30%,±40%, or ±50% of a given value. The value may be, e.g., 20 nm, 100 nm,500 nm, 1, 5, 10, 20, 50 microns, etc. The particles in any of theseembodiments can have any of the compositions described herein. Thepopulation can comprise particles having different compositions, in anyratio. The populations of particles may be used for any of the purposesdescribed herein, and methods for such use are an aspect of thisinvention.

Cross-linking reagents that can be used include but are not limited top-Azidobenzoyl hydrazide,N-(4-[p-Azidosalicyclamido]-butyl)-3′(2′-pyridyldithio)-propionamide,Bis(beta-[4-azidosalicylamido]-ethyl)disulfide,1,4-bismaleimidyl-2,3-diliydroxybutane, 1,6-Bismaleimidohexane,1,5-Difluoro-2,4-dinitrobenzene, Dimethyl adipimidate-2HCl, Dimethylsuberimidate-2HCl, Dimethyladipodimidate-2HCl,Dimethylpimelimidate-2HCl, Disuccinimidyl glutarate,Disuccinimidyltartrate, 1-Ethyl-3-[3-Dimethylanonopropyl]CarbodiimideHydrochloride, (N-Hydroxy succinimidyl)-4-Azidosalicylic acid,Sulfosuccinimdyl2-[7-azido-4-methyl-coumarin-3-acetamidomethyl-1,3-aminopropionate,N-Succinimidyl-iodoacetylaminobenzoate,N-Succinimidyl-3-[2-pyridylthio]propionate, and Succinimidyl6-[3-(2-pyridylathio)-propionamide]hexanoate (Pierce Chemical Co.,Rockford, Ill.) In one embodiment, the glucans are derivatized asdescribed in Nature Methods Vol 2 No. 11, p., 845, 2005, or a similarapproach. In one embodiment glucans are derivatized with a moiety thatprovides a free, reactive primary amine using a reagent such as2,6-diaminopyridine (DAP). The Schiff base azomethine can be reduced,e.g., by sodium cyanoborohydride to a stable secondary amine. In oneembodiment, the derivatized glucan is then reacted with anN-hydroxysuccinimide (NHS) ester, such as NHS-biotin.

Other crosslinking reagents comprise aldehyde, imide, cyano, halogen,carboxyl, activated carboxyl, anhydride and maleimide functional groups.In some embodiments, the cross-linking reagent may compriseheterobifunctional crosslinking reagents such as ABH, M2C2H, MPBH andPDPH (Pierce Chemical Co., Rockford, Ill.). See, e.g., Hermanson, G. T.(1996). Bioconjugate Techniques, Academic Press, Inc., for furtherdiscussion of cross-linking methods and reagents.

In another embodiment, conjugation of the glucan to the beads orparticles may be via use of beads comprising functional groups which canbe conjugated according to methods as disclosed by, e.g., Brumeanu etal. (Genetic Engineering News, Oct. 1, 1995, p. 16).

It is also possible to conjugate the beads/particles/solid support tothe glucan by non-covalent means. One convenient way for achievingnon-covalent conjugation comprises utilizing antibodies to the glucan,which are covalently or non-covalently attached to the particle, bead,etc. In another embodiment, non-covalent conjugation is achieved usingbiotin-avidin (where “avidin” should be understood to refer to any formof avidin). For example, avidin-coated or conjugated beads may becontacted with glucan derivatized with a biotin moiety.

In some embodiments, preparation of the conjugated glucans includespurification of the final conjugate substantially free of unconjugatedreactants. Purification may be achieved by affinity, gel filtration,hydrophobic chromatography, tangential ultrafiltration, diafiltration orion exchange chromatography based on the properties of either componentof the conjugate. For example, purification may reduce the amount of oneor more of the unconjugated reactants (e.g., glucan or solid support) to10% or less, 5% or less, or 1% or less of the amount of unconjugatedreactant that was originally present.

In some embodiments, the invention provides a particle comprising β 1-6glucan, which in some embodiments, is enriched for O-acetylated groups.In some embodiments, the particle comprises at least 50% β 1-6 glucan byweight. In some embodiments, the β1-6 glucan is homogeneouslydistributed in the particle. It is to be understood that the particlescomprising β 1-6 glucan of this invention, may in turn encompass anyembodiment appropriate thereto, as described herein.

In one embodiment, the conjugated glucan is enriched for O-acetylatedgroups, and in one embodiment, contains at least 25% by weightO-acetylated glucan, or any related embodiment as herein described. Inone embodiment, the glucan is conjugated to a microsphere, which in oneembodiment, has a diameter of about 1-100 microns. In one embodiment,the microsphere has a diameter which ranges from about 10-50 microns. Inanother embodiment, the microsphere has a diameter which ranges fromabout 5-40 microns. In another embodiment the diameter ranges from 0.1to 5 microns. In another embodiment the diameter ranges from 0.5 to 1micron. In another embodiment, the particle or bead is in the nanometerrange, e.g., 100 to 500 nm.

In one embodiment, the term “bead” or “particle” or “solid support”refers to a material, which is spherical. In another embodiment, term“bead” or “particle” or “solid support” refers to a material, which isnon-spherical. In one embodiment, non-spherical beads or particlespossess a longest axis or longest dimension between any two points ontheir surface within any of the afore-mentioned ranges. In oneembodiment, the dimensions of the particle (e.g., diameter) are selectedto promote phagocytosis of the particles by phagocytic cells, such asneutrophils, macrophages or dendritic cells.

In one embodiment, the term “bead” or “particle” or “solid support”refers to any solid or gelled, or sol-gel-based material, to which theglucan can be adhered, of a size and composition, which can be taken upby phagocytic cells.

In one embodiment, the kits/compositions of this invention comprise ormethods of this invention make use of beads or particles havingdimensions and surface density of glucan (e.g., β-1,6 glucan, optionallyenriched for O-acetylated groups), that is efficiently phagocytosed byantigen presenting cells as compared, e.g., with particles havingdifferent dimensions and/or surface density of glucan.

In one embodiment, conjugation to the solid support may be accomplishedwith a direct linkage via reaction with solid supports comprising areactive functional group.

Linkages via a linker group may be made using any known procedure, forexample, the procedures described, for example, in U.S. Pat. Nos.6,642,363; 4,882,317; or 4,695,624. A useful type of linkage is anadipic acid linker, which may be formed by coupling a free —NH₂ group onan aminated glucan with adipic acid (using, for example, diimideactivation), and then coupling a protein to the resultingsaccharide-adipic acid intermediate. Another type of linkage is acarbonyl linker, which may be formed by reaction of a free hydroxylgroup of a modified glucan with CDI followed by reaction with a proteinto form a carbamate linkage. Other linkers include B-propionamido,nitrophenyl-ethylamine, haloacyl halides, glycosidic linkages,6-aminocaproic acid, ADH, C4 to C 12 moieties, etc.

In another embodiment, the invention provides a particle comprising β1-6 glucan. In certain embodiments, the particle consists of at least10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99%β 1-6 glucan by dry weight. In certain embodiments, the particleconsists essentially of β 1-6 glucan. In certain embodiments, theparticle consists essentially of β 1-6 glucan, exclusive of any solventcomponent, such as water. In certain embodiments the β1-6 glucan isenriched for O-acetylated groups. In certain embodiments the particlecontains less than 50%, 40%, 30%, 20%, 10%, or 5% a 1-3 glucan by dryweight. The invention further provides a composition containing any ofthe afore-mentioned particles comprising or consisting essentially of β1-6 glucan, optionally enriched for O-acetylated groups. The compositionmay further contain a pharmaceutically acceptable carrier or adjuvant.The invention further provides a method of modulating the immuneresponse of a mammalian subject comprising administering any of theafore-mentioned particles, or a composition containing any of theafore-mentioned particles, to the subject. The particle can be preparedusing any method known in the art. The particles can be milled or sievedto achieve a desired size. In certain embodiments the β 1-6 glucan isdistributed evenly, or homogeneously, in the particle. In certainembodiments “distributed evenly” means that the β1-6 glucan is notencapsulated within a different material, does not simply coat thesurface of a particle comprised of a different material, or is notcovalently or non-covalently attached to the surface of a particlecomposed of a different material. Instead, in certain embodiments the β1-6 glucan, optionally mixed with another material, is formed into aparticle such that the β 1-6 glucan is located throughout substantiallythe entire volume of the particle. It will be appreciated that thedensity of the β 1-6 glucan may vary but will generally vary graduallyand continuously throughout the particle rather than abruptly.

In another embodiment, this invention provides a β1-6 glucan conjugatedto a solid support, wherein the solid support is a substrate, on or inwhich it is useful to conduct an assay. For example, and in someembodiments, the kits/methods of this invention make use of a microtiterplate, or 96 well plate to which a β 1-6 glucan has been adhered, and anassay is conducted within the wells/plate. Such substrates may compriseany suitable material, for example, being resistant to solvents, beingtransparent such that spectrophotometry may be conducted on the sampleswithin the substrate.

In some embodiments, the β 1-6 glucan for use in methods of thisinvention may further comprise a targeting moiety. In some embodiments,the targeting moiety is for a particular cell type, or in someembodiments, a diseased cell such as, for example, an infected cell, ora neoplastic cell or a preneoplastic cell. In some embodiments, forexample, targeting of a virally infected cell may be accomplished vialinkage of the glucan with a viral co-receptor. In some embodiments,targeting moieties may include integrins or class II molecules of theMHC, which may be upregulated on infected cells such as professionalantigen presenting cells.

In some embodiments, targeting of an infected cell results in enhancedtherapeutic responses to infection in the subject. For example, and insome embodiments, targeting the infected cell enhances phagocytosisand/or cytotoxic responses to the pathogen, or in some embodiments,enhances complement-mediated lysis of the pathogen. In some embodiments,targeting of the infected cell enhances the immune response to thepathogen.

In some embodiments, the targeting moiety specifically interacts with aneoplastic or preneoplastic cell, as described herein, and comprisingany embodiment thereof. In some embodiments, the use of a β1-6 glucanlinked to a targeting moiety, which targets a neoplastic orpreneoplastic cell promotes host anticancer responses. In someembodiments, such targeting promotes tumor cell lysis, or in someembodiments, enhances host anti-tumor responses.

In some embodiments, and without limitation, use of the glucans, β1-6glucan linked to a targeting moiety and/or compositions of thisinvention target the polysaccharide to an antigen expressed specificallyon cancer cells and thereby enhance complement-mediated lysis of thecells.

In one embodiment, this invention provides a method of stimulating animmune response in a subject, comprising administering to the subjectpurified β-1-6-glucan and an agent, which biases antibody production toyield relatively greater amounts of immunoglobulin G (IgG) 1, 2 or 3versus immunoglobulin G (IgG) 4.

According to this aspect of the invention, and in one embodiment, thesubject is administered the purified β-1-6-glucan and the agentconcurrently, or in another embodiment the subject is administeredpurified β-1,6-glucan and the agent sequentially. In another embodimentthe subject is administered said purified β-1-6-glucan and said cytokineeach at least two times.

In one embodiment, the agent is a cytokine, which in one embodiment isinterleukin-2, interleukin-12 or interferon-γ or a combination thereof.In another embodiment, the agent downmodulates interleukin-4 orinterleukin-10 production or interferes with interleukin-4 orinterleukin-10 activity.

In one embodiment, the subject is further exposed to an antigenassociated with a target of the immune response, which in one embodimentis a tumor-associated antigen. According to this aspect of theinvention, and in one embodiment, the subject has a hyperplastic orpreneoplastic lesion, and in another embodiment, the method treats,delays progression of, prolongs remission of, or reduces the incidenceor severity of cancer in the subject. In another embodiment, the subjecthas cancer. In another embodiment, the subject has not been diagnosedwith cancer. In another embodiment, the subject has not been diagnosedwith a tumor.

In another embodiment, the antigen is derived from a pathogen, which inone embodiment is a fungus. In one embodiment, the method treats, delaysprogression of, prolongs latency of, or reduces the incidence orseverity of infection in the subject.

In some embodiments, the method comprising targeting the 0 glucan andoptionally the cytokine to neoplastic or preneoplastic cells or tissue,or tumors, which can be accomplished by targeting a tumor antigen, asherein described. In some embodiments, such cells may expressadrenomedullin receptors (ADMR), a calcitonin receptor-like receptor(CRLR), CDI 17 or any combination of tumor associated antigens, asherein described.

According to this aspect, and in one embodiment, by targeting cellsexpressing adrenomedullin receptors with the linked glucans of thisinvention, lung, pancreas, ovary, and other related cancers may betreated. In some embodiments, by targeting cells expressing CRLR and/orCDI 17, with the linked glucans of this invention, vascular tumors,gliomas, and/or other related cancers may be treated.

In some embodiments, reference herein to a targeting moiety is to beunderstood to encompass an antibody, or fragment thereof as describedherein, a naturally occurring peptide ligand for the referencedreceptor, or a modified form thereof, such as, for example, a truncationproduct. In some embodiments, reference herein to a targeting moiety isto be understood to encompass artificial peptides, small molecules, andthe like.

In some embodiments, this invention provides for the use of the glucans,β 1-6 glucans linked to a targeting moiety and/or compositions of thisinvention (as described herein, including any embodiment thereof) as ameans to determine neoplastic or preneoplastic cell or tissueresponsiveness to a treatment regimen. In some embodiments, such methodincludes obtaining a tumor sample from the subject or biopsy materialcontaining the neoplastic or preneoplastic cells and assessing thesensitivity or resistance of the cells to in vitro lysis and/ordetermining the level of expression and/or secretion of an endogenouscomplement control protein.

In some embodiments the tumor cell expresses or overexpresses (e.g.,relative to a normal cell of the cell type or tissue of origin of thatcell) an endogenous complement control protein such as complementreceptor 1 (CR1 or CD35), decay-accelerating factor (DAF or CD55),membrane cofactor protein (MCP of CD46), complement factor H (fH) (orFHL-I) and/or C4b-binding protein (C4BP).

In some embodiments, this invention provides for the use of the glucans,β 1-6 glucans linked to a targeting moiety and/or compositions of thisinvention (as described herein, including any embodiment thereof) as ameans to target pathological vasculature, such as, for example,atherosclerotic vasculature, or in some embodiments, targetingpathologic neo-vasculature such as tumor-associated neovasculature forpurposes of enhancing elimination of such vasculature.

According to this aspect of the invention and in some embodiments, thetargeting moiety comprises, inter alia, an antibody or antibody fragmentor ligand specifically interacting with a component of such vascuature,for example, an agent specifically interacting with VEGF, tissue factor,a clotting factor, vascular cell adhesion molecules, integrins,selectins, or any other marker expressed on or at the surface ofendothelial cells.

In one embodiment, the targeting moiety is a peptide, an antibody, anantibody fragment, a receptor, Protein A, Protein G, biotin, avidin,streptavidin, a metal ion chelate, an enzyme cofactor, a nucleic acid ora ligand.

In some embodiments, such a targeting moiety may comprise an antibody orantibody fragment. In some embodiments, such antibody or antibodyfragment will specifically interact with a desired target, as describedherein, and linkage of said antibody or fragment with the glucan doesnot inhibit such interaction.

In some embodiments, the term “antibody” refers to intact molecules aswell as functional fragments thereof, such as Fab, F(ab′)2, and Fv thatare capable of specifically interacting with a desired target, asdescribed herein. In some embodiments, the antibody fragments comprise:

(1) Fab, the fragment which contains a monovalent antigen-bindingfragment of an antibody molecule, which can be produced by digestion ofwhole antibody with the enzyme papain to yield an intact light chain anda portion of one heavy chain;

(2) Fab′, the fragment of an antibody molecule that can be obtained bytreating whole antibody with pepsin, followed by reduction, to yield anintact light chain and a portion of the heavy chain; two Fab′ fragmentsare obtained per antibody molecule; (3) (Fab′)₂, the fragment of theantibody that can be obtained by treating whole antibody with the enzymepepsin without subsequent reduction; F(ab′)2 is a dimer of two Fab′fragments held together by two disulfide bonds;

(4) Fv, a genetically engineered fragment containing the variable regionof the light chain and the variable region of the heavy chain expressedas two chains; and

(5) Single chain antibody (“SCA”), a genetically engineered moleculecontaining the variable region of the light chain and the variableregion of the heavy chain, linked by a suitable polypeptide linker as agenetically fused single chain molecule.

Methods of making these fragments are known in the art. (See forexample, Harlow and Lane, Antibodies: A Laboratory Manual, Cold SpringHarbor Laboratory, New York, 1988, incorporated herein by reference).

In some embodiments, the antibody fragments may be prepared byproteolytic hydrolysis of the antibody or by expression in E. coli ormammalian cells (e.g., Chinese hamster ovary cell culture or otherprotein expression systems) of DNA encoding the fragment.

Antibody fragments can, in some embodiments, be obtained by pepsin orpapain digestion of whole antibodies by conventional methods. Forexample, antibody fragments can be produced by enzymatic cleavage ofantibodies with pepsin to provide a 5 S fragment denoted F(ab′)2. Thisfragment can be further cleaved using a thiol reducing agent, andoptionally a blocking group for the sulfhydryl groups resulting fromcleavage of disulfide linkages, to produce 3.5S Fab′ monovalentfragments. Alternatively, an enzymatic cleavage using pepsin producestwo monovalent Fab′ fragments and an Fc fragment directly. These methodsare described, for example, by Goldenberg. U.S. Pat. Nos. 4,036,945 and4,331,647, and references contained therein, which patents are herebyincorporated by reference in their entirety. See also Porter, R. R.,Biochem. J., 73: 119-126, 1959. Other methods of cleaving antibodies,such as separation of heavy chains to form monovalent light-heavy chainfragments, further cleavage of fragments, or other enzymatic, chemical,or genetic techniques may also be used, so long as the fragments bind tothe antigen that is recognized by the intact antibody.

Fv fragments comprise an association of VH and VL chains. Thisassociation may be noncovalent, as described in Inbar et al, Proc. Nat'lAcad. Sci. USA 69:2659-62, 1972. Alternatively, the variable chains canbe linked by an intermolecular disulfide bond or cross-linked bychemicals such as glutaraldehyde. Preferably, the Fv fragments compriseVH and VL chains connected by a peptide linker. These single-chainantigen binding proteins (sFv) are prepared by constructing a structuralgene comprising DNA sequences encoding the VH and VL domains connectedby an oligonucleotide. The structural gene is inserted into anexpression vector, which is subsequently introduced into a host cellsuch as E. coli. The recombinant host cells synthesize a singlepolypeptide chain with a linker peptide bridging the two V domains.Methods for producing sFvs are described, for example, by Whitlow andFilpula, Methods, 2: 97-105, 1991; Bird et al., Science 242:423-426,1988; Pack et al., Bio/Technology 11: 1271-77, 1993; and Ladner et al.,U.S. Pat. No. 4,946,778, which is hereby incorporated by reference inits entirety.

Another form of an antibody fragment is a peptide coding for a singlecomplementarity-determining region (CDR). CDR peptides (“minimalrecognition units”) can be obtained by constructing genes encoding theCDR of an antibody of interest. Such genes are prepared, for example, byusing the polymerase chain reaction to synthesize the variable regionfrom RNA of antibody-producing cells. See, for example, Larrick and Fry,Methods, 2: 106-10, 1991.

In some embodiments, the antibodies or fragments as described herein maycomprise “humanized forms” of antibodies. In some embodiments, the term“humanized forms of antibodies” refers to non-human (e.g., murine)antibodies, which are chimeric molecules of immunoglobulins,immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab′,F(ab′).sub.2 or other antigen-binding subsequences of antibodies) whichcontain minimal sequence derived from non-human immunoglobulin.Humanized antibodies include human immunoglobulins (recipient antibody)in which residues form a complementary determining region (CDR) of therecipient are replaced by residues from a CDR of a non-human species(donor antibody) such as mouse, rat or rabbit having the desiredspecificity, affinity and capacity. In some instances, Fv frameworkresidues of the human immunoglobulin are replaced by correspondingnon-human residues. Humanized antibodies may also comprise residueswhich are found neither in the recipient antibody nor in the importedCDR or framework sequences. In general, the humanized antibody willcomprise substantially all of at least one, and typically two, variabledomains, in which all or substantially all of the CDR regions correspondto those of a non-human immunoglobulin and all or substantially all ofthe FR regions are those of a human immunoglobulin consensus sequence.The humanized antibody optimally also will comprise at least a portionof an immunoglobulin constant region (Fc), typically that of a humanimmunoglobulin [Jones et al, Nature, 321:522-525 (1986); Riechmann etal., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol,2:593-596 (1992)].

Methods for humanizing non-human antibodies are well known in the art.Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues are often referred to as import residues, which aretypically taken from an import variable domain. Humanization can beessentially performed following the method of Winter and co-workers[Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)], bysubstituting rodent CDRs or CDR sequences for the correspondingsequences of a human antibody. Accordingly, such humanized antibodiesare chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantiallyless than an intact human variable domain has been substituted by thecorresponding sequence from a non-human species. In practice, humanizedantibodies are typically human antibodies in which some CDR residues andpossibly some FR residues are substituted by residues from analogoussites in rodent antibodies.

Human antibodies can also be produced using various techniques known inthe art, including phage display libraries [Hoogenboom and Winter, J.Mol. Biol, 227:381 (1991); Marks et al., J. Mol. Biol, 222:581 (1991)].The techniques of Cole et al. and Boerner et al. are also available forthe preparation of human monoclonal antibodies (Cole et al., MonoclonalAntibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985) and Boerner etal., J. Immunol, 147(1):86-95 (1991)]. Similarly, human can be made byintroducing of human immunoglobulin loci into transgenic animals, e.g.,mice in which the endogenous immunoglobulin genes have been partially orcompletely inactivated. Upon challenge, human antibody production isobserved, which closely resembles that seen in humans in all respects,including gene rearrangement, assembly, and antibody repertoire. Thisapproach is described, for example, in U.S. Pat. Nos. 5,545,807;5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016, and in thefollowing scientific publications: Marks et al, Bio/Technology 10,779-783 (1992); Lonberg et al., Nature 368 856-859 (1994); Morrison,Nature 368 812-13 (1994); Fishwild et al., Nature Biotechnology 14,845-51 (1996); Neuberger, Nature Biotechnology 14, 826 (1996); Lonbergand Huszar, Intern. Rev. Immunol. 13 65-93 (1995).

In one embodiment, the targeting moiety is an antibody or fragmentthereof, specifically recognizing a neutrophil, for example, andantibody specifically recognizing L-selectin, β2-integrins, complementreceptor 1 (CR-I), decay-accelerating factor (DAF), C5a receptor,intercellular adhesion molecule-1 (ICAM-1), ICAM-3 and others as will beappreciated by one skilled in the art.

In some embodiments, phagocytic cells are targeted by a moleculeinteracting with Fc receptors, chemokine receptors, CD40, CD80, CD86,MHC class II molecules, CD69, ADAM8, CD14, CD163, CD33, CD63, CD68,CD74, CHIT1, CHSTIO, CSFIR, DPP4, FABP4, FCGRIA, ICAM2, ILIR2, ITGAI,ITGA2, S100A8, TNFRSF8, and others as will be appreciated by one skilledin the art.

In another embodiment, the targeting moiety may be any appropriatemoiety, for example, aptamers, naturally occurring or artificialligands, or engineered binding proteins may comprise the targetingmoieties as described herein, and their physical association with aglucan as herein described can be readily accomplished by any number ofmeans known in the art, including, for example, the methods describedhereinbelow, or variations thereof, to suit the particular nature of thetargeting moiety chosen.

In one embodiment, the targeting moiety enhances attachment to the cell,or, in another embodiment, enhances homing to the cell. In oneembodiment, the targeting moiety enhances attachment following supply ofan energy source. In one embodiment, the targeting moiety is chemicallyattached to the glucan via a chemical cross-linking group, or in anotherembodiment, forms a stable association with the glucan, or, in anotherembodiment, forms an association with the glucan, which dissociatesfollowing changes in environmental conditions, such as, for example,salt concentration or pH.

In one embodiment, the targeting moiety may be an antibody, whichspecifically recognizes a molecule of interest, such as a protein ornucleic acid. In another embodiment, the antibody may specificallyrecognize a reporter molecule attached to a molecule of interest. Inanother embodiment, the targeting moiety may be an antibody fragment,Protein A, Protein G, biotin, avidin, streptavidin, a metal ion chelate,an enzyme cofactor, or a nucleic acid. In another embodiment, thetargeting moiety may be a receptor, which binds to a cognate ligand ofinterest, or associated with a cell or molecule of interest, or inanother embodiment, the targeting moiety may be a ligand which is usedto attach to a cell via interaction with its cognate receptor.

Linking the targeting moiety to the glucan of this invention may beaccomplished by any means known in the art, for example as describedfurther herein in Example 7, or for example, as described in U.S. Pat.No. 5,965,714, or United States Patent Publication No. 20070141084, orSchneerson et al, Proc Natl Acad Sci USA. 2003 Jul. 22; 100(15):8945-50, Lees et al., Vaccine. 1996 February; 14 (3): 190-8, or viathe use of a cross-linking agent as described herein, or other methods,as will be appreciated by one skilled in the art.

In some embodiments, glycosylated antibodies are used and theβ-1,6-glucan is linked to the glycosylated residue of the antibody, orin another embodiment, linkages may be multiple and involve multiplesites on the antibody, or targeting moiety, as will be understood by oneskilled in the art.

In some embodiments, linking the glucan to a targeting moiety results inenhanced phagocytosis and/or killing of the targeted cell or organism.In some embodiments, such lysis may be mediated by any professionalantigen presenting cell or killer cell, such as, for example,neutrophils, macrophages, dendritic cells, natural killer cells,cytotoxic T lymphocytes, and others.

In some embodiments, any O-acetylated glucan may be physicallyassociated with a targeting moiety, and comprise the glucans orcompositions of this invention, representing an embodiment thereof. Useof such O-acetylated glucans, for example β-1,3-glucans which have beenO-acetylated, for modulating immune responses, treating cancer orprecancerous lesions, promoting resolution of infection, or any methodas herein described is to be considered as part of this invention.

In some embodiments, any of the glucan preparations of the kits and foruse in the methods of this invention may be linked to a labeling agent,such that detection of the glucan is readily accomplished. In oneembodiment, the term “a labeling agent” refers to a molecule whichrenders readily detectable that which is contacted with a labelingagent. In one embodiment, the labeling agent is a marker polypeptide.The marker polypeptide may comprise, for example, green fluorescentprotein (GFP), DS-Red (red fluorescent protein), secreted alkalinephosphatase (SEAP), beta-galactosidase, luciferase, or any number ofother reporter proteins known to one skilled in the art. In anotherembodiment, the labeling agent may be conjugated to another moleculewhich provides greater specificity for the target to be labeled. Forexample, and in one embodiment, the labeling agent is a fluorochromeconjugated to an antibody which specifically binds to a given targetmolecule, or in another embodiment, which specifically binds anotherantibody bound to a target molecule, such as will be readily appreciatedby one skilled in the art. In some embodiments, the glucan linked to anantibody incorporates a fluorochrome in the antibody as will beappreciated by one skilled in the art.

In one embodiment, this invention provides for the combined use of, orcompositions comprising β-glucans and an adjuvant, or when β-glucanbased adjuvants are used, a second adjuvant is administered with theβ-glucan. In some embodiments, the adjuvant may include, but is notlimited to: (A) aluminium compounds (e.g., aluminium hydroxide,aluminium phosphate, aluminium hydroxyphosphate, oxyhydroxide,orthophosphate, sulphate, etc. [e.g., see chapters 8 & 9 of ref. 96]),or mixtures of different aluminium compounds, with the compounds takingany suitable form (e.g., gel, crystalline, amorphous, etc.), and withadsorption being preferred; (B) MF59 (5% Squalene, 0.5% Tween 80, and0.5% Span 85, formulated into submicron particles using amicrofluidizer); (C) liposomes; (D) ISCOMs, which may be devoid ofadditional detergent; (E) SAF, containing 10% Squalane, 0.4% Tween 80,5% pluronic-block polymer L121, and thr-MDP, either micro fluidized intoa submicron emulsion or vortexed to generate a larger particle sizeemulsion; (F) Ribi™ adjuvant system (RAS), (Ribi Immunochem) containing2% Squalene, 0.2% Tween 80, and one or more bacterial cell wallcomponents from the group consisting of monophosphorylipid A (MPL),trehalose dimycolate (TDM), and cell wall skeleton (CWS), preferablyMPL+CWS (Detox™); (G) saponin adjuvants, such as Qui1A or QS21, alsoknown as Stimulon™; (H) chitosan; (I) complete Freund's adjuvant (CFA)and incomplete Freund's adjuvant (IFA); (J) cytokines, such asinterleukins (e.g., IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, etc.),interferons (e.g., interferon-γ), macrophage colony stimulating factor,tumor necrosis factor, etc.; (K) monophosphoryl lipid A (MPL) or3-O-deacylated MPL (3dMPL)]; (L) combinations of 3dMPL with, forexample, QS21 and/or oil-in-water emulsions; (M) oligonucleotidescomprising CpG motifs]i.e., containing at least one CG dinucleotide,with 5-methylcytosine optionally being used in place of cytosine; (N) apolyoxyethylene ether or a polyoxyethylene ester; (O) a polyoxyethylenesorbitan ester surfactant in combination with an octoxynol or apolyoxyethylene alkyl ether or ester surfactant in combination with atleast one additional non-ionic surfactant such as an octoxynol; (P) animmuno-stimulatory oligonucleotide (e.g., a CpG oligonucleotide) and asaponin; (O) an immuno-stimulant and a particle of metal salt; (R) asaponin and an oil-in-water emulsion; (S) a saponin (e.g.,QS21)+3dMPL+IL12 (optionally+a sterol); (T) E. coli heat-labileenterotoxin (“LT”), or detoxified mutants thereof, such as the K63 orR72 mutants; (U) cholera toxin (“CT”), or diphtheria toxin (“DT”) ordetoxified mutants of either; (V) double-stranded RNA; (W)monophosphoryl lipid A mimics, such as aminoalkyl glucosaminidephosphate derivatives e.g., RC-529]; (X) polyphosphazene (PCPP); or (Y)a bioadhesive such as esterified hyaluronic acid microspheres or amucoadhesive such as crosslinked derivatives of poly(acrylic acid),polyvinyl alcohol, polyvinyl pyrollidone, polysaccharides andcarboxymethylcellulose.

Muramyl peptides include N-acetyl-muramyl-L-threonyl-D-isoglutamine(thr-MDP), N-acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP),N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)ethylamineMTP-PE), etc.

In another embodiment, this invention provides for the combined use ofJ-glucans, an agent, which biases antibody production to yieldrelatively greater amounts of immunoglobulin G (IgG) 1, 2 or 3 versusimmunoglobulin G (IgG) 4 and an antigen.

In various embodiments, the antigen may be any molecule recognized bythe immune system of the subject as foreign. For example, the antigenmay be any foreign molecule, such as a protein (including a modifiedprotein such as a glycoprotein, a mucoprotein, etc.), a nucleic acid, acarbohydrate, a proteoglycan, a lipid, a mucin molecule, or othersimilar molecule, including any combination thereof. The antigen may, inanother embodiment, be a cell or a part thereof, for example, a cellsurface molecule. In another embodiment, the antigen may derive from aninfectious virus, bacteria, fungi, or other organism (e.g., protists),or part thereof. These infectious organisms may be active, in oneembodiment or inactive, in another embodiment, which may beaccomplished, for example, through exposure to heat or removal of atleast one protein or gene required for replication of the organism. Inone embodiment, the antigenic protein or peptide is isolated, or inanother embodiment, synthesized.

In one embodiment, the term “antigen” refers to a substance such as aprotein, peptide, or any fragment which stimulates or enhances an immuneresponse, following exposure to or contact with the antigen. In oneembodiment, the antigen is a “danger” signal interpreted by the immunesystem of a subject as to initiate or enhance an immune response as aconsequence of the signal. In another embodiment, the antigen representsthe host's ability to distinguish the presence of a molecule which is“non-self.

In one embodiment, the antigen is derived from a pathogen, an infectedcell, a neoplastic or preneoplastic cell. In another embodiment, theantigen is an autoantigen, or a molecule which initiates or enhances anautoimmune response.

In one embodiment, the antigen is derived from a parasitic agent, whichresides intracellularly during at least some stages of its life cycle.The intracellular parasites contemplated include for example, protozoa.Protozoa, which infect cells, include: parasites of the genus Plasmodium(e.g., Plasmodium falciparum, P. Vivax, P. ovale and P. malariae),Trypanosoma, Toxoplasma, Leishmania, Schislosoma, and Cryptosporidium.In another embodiment the parasitic agent resides extracellularly duringat least part of its life cycle. Examples include nematodes, trematodes(flukes), and cestodes. In some embodiments, the antigen is derived frombyproducts of infection with the protozoa described, for example, eggantigens of the Schistosoma, antigens uniquely expressed from Toxoplasmacysts, and others, as will be appreciated by one skilled in the art.

In one embodiment, the antigen is derived from a diseased and/orabnormal cell. The diseased or abnormal cells contemplated include:infected cells, neoplastic cells, pre-neoplastic cells, inflammatoryfoci, benign tumors or polyps, cafe au lait spots, leukoplakia, otherskin moles, self-reactive cells, including T and/or NK cells, etc

In one embodiment, the antigen is derived from an infectious virusincluding, inter-alia: Retroviridae (e.g., human immunodeficiencyviruses, such as HIV-I (also referred to as HTLV-III, LAV orHTLV-III/LAV, or HIV-III; and other isolates, such as HIV-LP;Picornaviridae (e.g., polio viruses, hepatitis A virus; enteroviruses,human coxsackie viruses, rhinoviruses, echoviruses); Calciviridae (e.g.,strains that cause gastroenteritis); Togaviridae (e.g., equineencephalitis viruses, rubella viruses); Flaviridae (e.g., dengueviruses, encephalitis viruses, yellow fever viruses); Coronaviridae(e.g., coronaviruses); Rhabdoviridae (e.g., vesicular stomatitisviruses, rabies viruses); Filoviridae (e.g., ebola viruses);Paramyxoviridae (e.g., parainfluenza viruses, mumps virus, measlesvirus, respiratory syncytial virus); Orthomyxoviridae (e.g., influenzaviruses); Bungaviridae (e.g., Hantaan viruses, bunga viruses,phleboviruses and Nairo viruses); Arena viridae (hemorrhagic feverviruses); Reoviridae (erg., reoviruses, orbiviurses and rotaviruses);Birnaviridae; Hepadnaviridae (Hepatitis B virus); Parvoviridae(parvoviruses); Papovaviridae (papilloma viruses, polyoma viruses);Adenoviridae (most adenoviruses); Herpesviridae (herpes simplex virus(HSV) 1 and 2, varicella zoster virus, cytomegalovirus (CMV), herpesviruses); Poxyiridae (variola viruses, vaccinia viruses, pox viruses);and Iridoviridae (e.g., African swine fever virus); and unclassifiedviruses (e.g., the etiological agents of Spongiform encephalopathies,the agent of delta hepatitis (thought to be a defective satellite ofhepatitis B virus), the agents of non-A, non-B hepatitis (class1=internally transmitted; class 2=parenterally transmitted (i.e.,Hepatitis C); Norwalk and related viruses, and astroviruses).

In one embodiment, the antigen is derived from bacteria including,inter-alia: Helicobacter pylori, Borellia burgdorferi, Legionellapneumophilia, Mycobacleria sps (e.g., M. tuberculosis, M. avium, M.intracellular, M. kansaii, M. gordonae), Staphylococcus aureus,Neisseria gonorrhoeae, Neisseria meningitidis, Listeria monocytogenes,Streptococcus pyogenes (Group A Streptococctus), Streptococcusagalactiae (Group B Streptococcus). Streptococcus (viridans group),Streptococcus faecalis, Streptococcus hovis, Streptococcus (anaerobicsps.), Streptococcus pneumonie, pathogenic Campylobacter sp.,Enterococcus sp. Chlamydia sp., Haemophilus influenzae, Bacillusantracis, corynebacterium diphtheriae, corynehacterium sp.,Erysipelothrix rhusiopathiae, Clostridium perfringers, Clostridiumtetani, Enterobacter aerogenes, Klebsiella pneumoniae, Pasturellamultocida, Bacteroides sp. Fusobacterium nucleatum, Streptobacillusmoniliformis, Treponema pallidium, Trepnema pertenue, Leptospira,Actinomyces israelii and Francisella tularensis.

In one embodiment, the antigen is derived from fungi, including,inter-alia: Absidia, such as Absidia corymbfera, Ajellomyces, such asAjellomyces capsulatus, Ajellomyces dermatitidis, Arthroderma, such asArthroderma benhamiae, Arthnurerma fulvum, Arthroderma gypseum,Arthroderma incurvatum, Arthroderma otae, Arthroderma vanbreuseghemii,Aspergillus, such as Aspergillus flavus, Aspergillus fumigatus,Aspergillus niger, Blastomyces, such as Blastomyces dermatitidis,Candida, such as Candida albicans, Candida glabrata, Candidaguilliermondii, Candida krusei, Candida parapsilosis, Candidatropicalis, Candida pelliculosa Cladophialophora, such asCladophialophora carrionii, Coccidioides, such as Coccidioides immitis,Cryptococcus, such as Cryptococcus neoformans, Cunninghamella,Epidermophyton, such as Epidermophyton floccosum, Exophiala, suchExophiala dermatitidis, Filobasidiella, such as Filobasidiellaneofarmans. Fonsecaea, such as Fonsecaea pedrosoi, Fusarium, such asFusarium solani, Geotrichum, such as Geotrichum candidum, Histoplasma,such as Histoplama capsulatum, Hortaea, such as Hortaea werneckii,Issatschenkia, such as Issatschenkia orientalis, Madurella, suchMadurella grisae, Malassezia, such as Malassezia furfur, Malasseziaglobosa, Malassezia obtuse, Malassezia pachydermatis, Malasseziarestricla, Malassezia sloofiae, Malassezia sympodialis, Microsporum,such as Microsporum canis, Microsporum fulvum, Microsporum gypseum,Mucor, such as Mucor circinelloides, Nectria, such as Nectriahaematococca, Paecilomyces, such as Paecilomnyces variotii,Paracoccidioides, such as Paracoccidioides brasiliensis, Penicillium,such as Penicillium marneffei, Pichia, such as Pichia anomala, Pichiaguilliermondii, Pneumocystis, such as Pneumocystis carinii,Pseudallescheria, such as Pseudallescheria boydii, Rhizopus, such asRhizopus oryzae, Rhodotorula, such as Rhodotorula rubra, Scedosporium,such as Scedosporiumn apiospermum, Schizophyllum, such as Schizophyllumcommune, Sporothrix, such as Sporothrix schenckii, Trichophyton, such asTrichophyton mentagrophytes, Trichophyton rubrum, Trichophytonverrucosum, Trichophyton violaceum, Trichosporon, such as Trichosporonasahii, Trichtosporon cutaneum, Trichosporon inkin, Trichosporonmucoides, or others.

In one embodiment, the pathogenic fungus infects human hosts. In oneembodiment the pathogenic fungus infects non-human animals.

In some embodiments, the compositions and methods of this inventionallow for the combined use of multiple antigens from the same source,multiple antigens from the same class of organism, multiple antigensfrom different classes of organisms, or any combination thereof.

In another embodiment, this invention provides a method of treating,delaying progression of, or reducing the incidence or severity of aninfection in a subject, said method comprising administering to saidsubject a composition comprising purified β 1-6 glucan. In certainembodiments of the invention the infection is one due to a pathogenicfungus. In certain embodiments of the invention the infection is one dueto a pathogenic bacterium, virus, or parasite. In certain embodiments ofthe invention the subject receives, in addition to a composition of thisinvention, any agent known in the art to be useful for treating orpreventing an infection from which the subject is at risk from which thesubject suffers. Thus in one embodiment the method comprisesadministering to a subject (i) a composition of this inventioncomprising β 1-6 glucan; and (ii) a known anti-fungal, anti-bacterial,anti-viral, or anti-parasitic agent. The composition and anti-fungalagent could be administered in a single composition or separately. Insome embodiments, such separate administration may be within up to 24 orup to 48 hours apart, and in some embodiments, less than an hour apart.The composition could be suitable for use in humans, for veterinaryapplications, or both.

In some embodiments, the use of S glucan and optionally an agent, whichbiases antibody production to yield relatively greater amounts ofimmunoglobulin G (IgG) 1, 2 or 3 versus immunoglobulin G (IgG) 4 thereofof this invention stimulate, enhance or facilitate complement fixation.In some embodiments, this effect is antibody-mediated.

According to this aspect, and in some embodiments, the use of 1 glucanand optionally an agent, which biases antibody production to yieldrelatively greater amounts of immunoglobulin G (IgG) 1, 2 or 3 versusimmunoglobulin G (IgG) 4 thereof of this invention stimulates, enhancesor promotes immune responses, which involve complement fixation,resulting in therapeutic effects in the subject. In some embodiments,such use may be directed to treating sepsis in the subject. In someembodiments, such use may be directed to treating Chagas disease in asubject, a pulmonary pathogen, or a parasite or helminth. In someembodiments, such use is directed to treating a viral infection, such asHSV.

In some embodiments, the methods according to this aspect of theinvention may further comprise administration of an agent which promoteselaboration of the complement cascade. In some embodiments, according tothis aspect of the invention, the methods may further compriseadministration of an antibody which specifically recognizes thepathogenic agent with which the subject is infected.

In another embodiment, the method of stimulating an immune response ofthis invention is directed to stimulating an anticancer response. In oneembodiment, the method further comprises exposing the subject to anantigen, which is a tumor-associated antigen. In one embodiment, thesubject has a hyperplastic or preneoplastic lesion. In anotherembodiment, the subject has cancer.

In one embodiment, cancers associated with the following cancer antigenmay be treated or prevented by the methods and compositions of theinvention. KS 1/4 pan-carcinoma antigen (Perez and Walker, 1990, J.Immunol. 142:32-37; Bumal, 1988, Hybridoma 7(4):407-415), ovariancarcinoma antigen (CAA125) (Yu et al, 1991, Cancer Res. 51(2):48-475),prostatic acid phosphate (Tailor et al., 1990, Nucl. Acids Res.18(1):4928), prostate specific antigen (Henttu and Vihko, 1989, Biochem.Biophys. Res. Comm. 10(2):903-910; Israeli et al, 1993, Cancer Res.53:227-230), melanoma-associated antigen p97 (Estin et al, 1989, J.Natl. Cancer Instit 81 (6):445-44), melanoma antigen gp75 (Vijayasardahlet al, 1990, J. Exp. Med. 171(4): 1375-1380), high molecular weightmelanoma antigen (HMW-MAA) (Natali et al, 1987, Cancer 59:55-3;Mittelman et al, 1990, J. Clin. Invest. 86:2136-2144)), prostatespecific membrane antigen, carcinoembryonic antigen (CEA) (Foon et al,1994, Proc. Am. Soc. Clin. Oncol. 13:294), polymorphic epithelial mucinantigen, human milk fat globule antigen, Colorectal tumor-associatedantigens such as: CEA, TAG-72 (Yokata et al, 1992, Cancer Res.52:3402-3408), CO17-1A (Ragnhammar et al, 1993, Int. J. Cancer53:751-758); GICA 19-9 (Herlyn et al, 1982, J. Clin. Immunol. 2:135),CTA-1 and LEA, Burkitt's lymphoma antigen-38.13, CD19 (Ghetie et al,1994, Blood 83:1329-1336), human B-lymphoma antigen-CD20 (Reffet al,1994, Blood 83:435-445), CD33 (Sgouros et al, 1993, J. Nucl. Med.34:422-430), melanoma specific antigens such as ganglioside GD2 (Salehet al., 1993, J. Immunol, 151, 3390-3398), ganglioside GD3 (Shitara etal., 1993, Cancer Immunol. Immunother. 36:373-380), ganglioside GM2(Livingston et al., 1994, J. Clin. Oncol. 12:1036-1044), ganglioside GM3(Hoon et al., 1993, Cancer Res. 53:5244-5250), tumor-specifictransplantation type of cell-surface antigen (TSTA) such asvirally-induced tumor antigens including T-antigen DNA tumor viruses andenvelope antigens of RNA tumor viruses, oncofetalantigen-alpha-fetoprotein such as CEA of colon, bladder tumor oncofetalantigen (Hellstrom et al., 1985, Cancer. Res. 45:2210-2188),differentiation antigen such as human lung carcinoma antigen L6, L20(Hellstrom et al., 1986, Cancer Res. 46:3917-3923), antigens offibrosarcoma, human leukemia T cell antigen-Gp37(Bhattacharya-Chatterjee et al., 1988, J. of Immun. 141:1398-1403),neoglycoprotein, sphingolipids, breast cancer antigen such as EGFR(Epidermal growth factor receptor), HER2 antigen (p185HER2), polymorphicepithelial mucin (PEM) (Hilkens et al., 1992, Trends in Bio. Chem. Sci.17:359), malignant human lymphocyte antigen-APO-1 (Bernhard et al.,1989, Science 245:301-304), differentiation antigen (Feizi, 1985, Nature314:53-57) such as I antigen found in fetal erythrocytes and primaryendoderm, 1(Ma) found in gastric adenocarcinomas, M18 and M39 found inbreast epithelium, SSEA-1 found in myeloid cells, VEP8, VEP9, MyI,VIM-D5, and D156-22 found in colorectal cancer, TRA-1-85 (blood groupH), C 14 found in colonic adenocarcinoma, F3 found in lungadenocarcinoma, AH6 found in gastric cancer, Y hapten, Ley found inembryonal carcinoma cells, TL5 (blood group A), EGF receptor found inA431 cells, E1 series (blood group B) found in pancreatic cancer, FC10.2 found in embryonal carcinoma cells, gastric adenocarcinoma, CO-514(blood group Lea) found in adenocarcinoma, NS-10 found inadenocarcinomas, CO-43 (blood group Leb), G49, EGF receptor, (bloodgroup ALeb/Ley) found in colonic adenocarcinoma, 19.9 found in coloncancer, gastric cancer mucins, TSA7 found in myeloid cells, R24 found inmelanoma, 4.2, GD3, D1.1, OFA-I, GM2, OFA-2, GD2, M1:22:25:8 found inembryonal carcinoma cells and SSEA-3, SSEA-4 found in 4-8-cell stageembryos. In another embodiment, the antigen is a T cell receptor derivedpeptide from a cutaneous T cell lymphoma (see Edelson, 1998, The CancerJournal 4:62).

In another embodiment, the antigen is derived from HER2/neu orchorio-embryonic antigen (CEA) for suppression/inhibition of cancers ofthe breast, ovary, pancreas, colon, prostate, and lung, which expressthese antigens. Similarly, mucin-type antigens such as MUC-1 can be usedagainst various carcinomas; the MAGE, BAGE, and Mart-1 antigens can beused against melanomas. In one embodiment, the methods may be tailoredto a specific cancer patient, such that the choice of antigenic peptideor protein is based on which antigen(s) are expressed in the patient'scancer cells, which may be predetermined by, in other embodiments,surgical biopsy or blood cell sample followed by immunohistochemistry.

In another embodiment, this invention provides for the combined use ofβ-glucans and an agent, which biases antibody production to yieldrelatively greater amounts of immunoglobulin G (IgG) 1, 2 or 3 versusimmunoglobulin G (IgG) 4 and optionally additional immunomodulatorycompounds.

Examples of useful agents include cytokines, chemokines, complementcomponents, immune system accessory and adhesion molecules and theirreceptors of human or non-human animal specificity. Useful examplesinclude, but are not limited to: GM-CSF, IL-2, IL-12, OX40, OX40L(gp34), lymphotactin, CD40, and CD40L. Further useful examples include,but are not limited to: interleukins for example interleukins 1 to 15,interferons alpha, beta or gamma, tumor necrosis factor,granulocyte-macrophage colony stimulating factor (GM-CSF), macrophagecolony stimulating factor (M-CSF), granulocyte colony stimulating factor(G-CSF), chemokines such as neutrophil activating protein (NAP),macrophage chemoattractant and activating factor (MCAF), RANTES,macrophage inflammatory peptides MIP-Ia and MIP-Ib, complementcomponents and their receptors, or an accessory molecule such as B7.1,B7.2, TRAP, ICAM-1,2 or 3 and cytokine receptors. OX40 and OX40-ligand(gp34) are further useful examples of immuno-modulatory proteins. It isto be understood that any agent, which stimulates the immune response toand in the process participates in the biasing, or facilitates theactivation of the biased response, in concert with the glucans as hereindescribed in a given immune response may be used in accordance with themethods of this invention, and is to be considered an embodimentthereof.

In one embodiment, uses according to this invention, may also compriseadministering an additional therapeutic, which may comprise ananti-inflammatory agent such as betamethasone, prednisolone, piroxicam,aspirin, flurbiprofen and(+)-N-{4-[3-(4-fluorophenoxy)phenoxy]-2-cyclopenten-1-yl}-N-hyroxyurea;an antiviral such as acyclovir, nelfinavir, or virazole; an antibioticsuch as ampicillin and penicillin G or belonging to the family ofpenicillins, cephalosporins, aminoglycosidics, macrolides, carbapenemand penem, beta-lactam monocyclic, inhibitors of beta-lactamases,tetracycline, polipeptidic antibiotics, chloramphenicol and derivatives,fusidic acid, lincomicyn, novobiocine, spectinomycin, poly-ethericionophores, quinolones: an anti-infective such as benzalkonium chlorideor chlorhexidine; dapsone, chloramphenicol, neomycin, cefaclor,cefadroxil, cephalexin, cephradine erythromycin, clindamycin,lincomycin, amoxicillin, ampicillin, bacampicillin, carbenicillin,dicloxacillin, cyclacillin, picloxacillin, hetacillin, methicillin,nafcillin, oxacillin, penicillin including penicillin G and penicillinV, ticarcillin rifampin and tetracycline; an antiinflammatory such asdiflunisal, ibuprofen, indomethacin, meclofenamate, mefenamic acid,naproxen, oxyphenbutazone, phenylbutazone, piroxicam, sulindac,tolmetin, aspirin and salicylates; antifungal such as amphotericin B,glucan synthesis inhibitors such as caspofungin, micafungin, oranidulafungin (LY303366), econazole, terconazole, fluconazole,voriconazole or griseofulvin; an antiprotozoal such as metronidazole; animidazole-type anti-neoplastic such as tubulazole; an anthelmintic agentsuch as thiabendazole or oxfendazole; an antihistamine such asastemizole, levocabastine, cetirizine, or cinnarizine; a decongestantsuch as pseudoephedrine; antipsychotics such as fluspirilene,penfluridole, risperidone or ziprasidone; an antineoplastic agent suchas platinum compounds (e.g., spiroplatin, cisplatin, and carboplatin),methotrexate, fluorouracil, adriamycin, mitomycin, ansamitocin,bleomycin, cytosine arabinoside, arabinosyl adenine, mercaptopolylysine,vincristine, busulfan, chlorambucil, melphalan (e.g., PAM, L-PAM orphenylalanine mustard), mercaptopurine, mitotane, procarbazinehydrochloride dactinomycin (actinomycin D), daunorubicin hydrochloride,doxorubicin hydrochloride, paclitaxel and other taxenes, rapamycin,manumycin A, TNP-470, plicamycin (mithramycin), aminoglutethimide,estramustine phosphate sodium, flutamide, leuprolide acetate, megestrolacetate, tamoxifen citrate, testolactone, trilostane, amsacrine(m-AMSA), asparaginase (L-asparaginase) Erwina asparaginase, interferon.alpha.-2a, interferon .alpha.-2b, teniposide (VM-26). vinblastinesulfate (VLB), vincristine sulfate, bleomycin sulfate, hydroxyureaprocarbazine, and dacarbazine; a mitotic inhibitor such as etoposide,colchicine, and the vinca alkaloids, a radiopharmaceutical such asradioactive iodine and phosphorus product, or any combination thereof.

In some embodiments, the methods of this invention are used totherapeutically or prophylactically treat animals or humans who are at aheightened risk of infection due to imminent surgery, injury, illness,radiation or chemotherapy, or other condition which deleteriouslyaffects the immune system. In some embodiments, the methods of thisinvention are used to treat patients who have a disease or disorderwhich causes the normal immune response to be reduced or depressed, suchas HIV infection (AIDS) or who are receiving immunosuppressive therapy(e.g., individuals who are transplant candidates or have received atransplant, individuals suffering from an autoimmune disease, etc.). Insome embodiments, the methods of this invention are used to pre-initiatean immune response in patients who are undergoing chemotherapy orradiation therapy, or who are at a heightened risk for developingsecondary infections or postoperative complications because of adisease, disorder or treatment resulting in a reduced ability tomobilize the body's normal responses to infection.

In one embodiment, stimulating said immune response comprisesstimulating an antigen-specific response

In some embodiments, use of the glucans as described herein, and methodsas herein described may function to enhance complement-mediated lysis ina subject. In some embodiments, such enhancement may involve thephagocytic cell response, for example, enhancing neutrophil ormacrophage, or other professional antigen presenting cell phagocytosisand cytotoxic responses. In some embodiments, such enhancement may beindependent of phagocytic cell involvement, for example, by enhancingmembrane attack complex formation and/or activity.

It is to be understood that the methods of this invention which bystimulating an immune response, in turn prevent disease, and/orameliorate disease, and/or alter disease progression are to beconsidered as part of this invention.

In some embodiments, the term “contacting” or “administering” refers toboth direct and indirect exposure to the indicated material.

In some embodiments, the methods of this invention make use of anon-sterile or sterile carrier or carriers for administration of thedescribed agents to cells, tissues or organisms, such as apharmaceutical carrier suitable for administration to an individual.Such carriers may include, but are not limited to, saline, bufferedsaline, dextrose, water, glycerol, and combinations thereof. Theformulation should suit the mode of administration.

The agents may be administered in any effective, convenient mannerincluding, for instance, administration by intravascular (i.v.),intramuscular (i.m.), intranasal (i.n.), subcutaneous (s.c), oral,rectal, intravaginal delivery, or by any means in which theglucan/composition can be delivered to tissue (e.g., needle orcatheter). Alternatively, topical administration may be desired forinsertion into epithelial cells. Another method of administration is viaaspiration or aerosol formulation. In some embodiments the methodscomprise administration of the described agents/glucans by implanting orintroducing into the body of a subject, an implant or other medical orsurgical device that comprises the glucan, e.g., as a component of acoating layer.

In one embodiment, the invention provides a food supplement comprising β1-6 glucan and/or other agents described herein.

In some embodiments, a food or food product is any substance that issubstantially non-toxic that can be metabolized by an organism to giveenergy and build tissue. In some embodiments, a food or food productdenotes a product intended for ingestion by a mammal, e.g, by humans,which has nutritional value. In some embodiments a food or food productdenotes a product regulated as a food or food product by the U.S. Foodand Drug Administration (FDA). In some embodiments, a food or foodproduct is a product packaged in a container bearing a label indicatingthat the product is a food or food product. In some embodiments, a foodor food product is a product packaged in a container bearing a labelproviding nutritional information regarding the product, such as thecalorie, fat, or protein content, or the content of one or more vitaminsor minerals. In some embodiments a food supplement (also referred to asa “dietary supplement”) is any substance that is added to a food or foodproduct. In some embodiments the food supplement comprises, in additionto a glucan of this invention, one or more essential nutrients, such asvitamins, minerals, and protein. In some embodiments, the foodsupplement is any product intended for ingestion as a supplement to thediet and may comprise, in addition to a glucan of this invention, one ormore vitamins, minerals, herbs, botanicals, and other plant-derivedsubstances; amino acids; and concentrates, metabolites, constituents andextracts of these substances. In some embodiments, the food, foodproduct, food supplement, or cosmetic composition is not intended todiagnose, cure, mitigate, treat, or prevent disease. In someembodiments, the food supplement is provided in a container or otherpackaging material labeled to indicate that the contents are a food ordietary supplement, e.g., in accordance with then current U.S. lawand/or FDA guidelines. In some embodiments, the food supplement orproduct comprises about 0.01 to 30 w/w % of the glucan, and mayadditionally comprise vitamins, oligosaccharides, dietary ingredients,proteins, or a combination thereof.

In some embodiments, the ratio of the components is not fixed, or inother embodiments, such ratio may range from about 0.01 to 30 w/w % per100 w/w %. Examples of food comprising aforementioned glucan of thisinvention therein are various food, beverage, gum, vitamin complex,health improving food and the like.

The composition may additionally comprise one or more than one oforganic acid, such as citric acid, fumaric acid, adipic acid, lacticacid, malic acid; phosphate, such as phosphate, sodium phosphate,potassium phosphate, acid pyrophosphate, polyphosphate; naturalanti-oxidants, such as polyphenol, catechin, alpha-tocopherol, rosemaryextract, vitamin C, licorice root extract, chitosan, tannic acid, phyticacid etc.

In some embodiments, the agents/glucans administered as part of themethods of this invention are formulated as a topical ointment, lotion,gel, or cream containing the active ingredient(s) in an amount of, forexample, 0.0001 to 50% w/w, e.g., 0.075 to 20% w/w (including activeingredient(s) in a range between 0.1% and 20% in increments of 0.1% w/wsuch as 0.6% w/w, 0.7% w/w, etc.), often 0.2 to 15% w/w and most often0.5 to 10% w/w). In some embodiments, when formulated in an ointment,the active ingredients may be employed with either a paraffinic or awater-miscible ointment base. In some embodiments, the activeingredients may be formulated in a cream with an oil-in-water creambase.

In some embodiments, the aqueous phase of the cream base may include,for example, at least 30% w/w of a polyhydric alcohol, i.e., an alcoholhaving two or more hydroxyl groups such as propylene glycol, butane1,3-diol, mannitol, sorbitol, glycerol and polyethylene glycol(including PEG 400) and mixtures thereof. In some embodiments, thetopical formulations may include a compound that enhances absorption orpenetration of the active ingredient(s) through the skin or otheraffected areas. Examples of such dermal penetration enhancers includedimethyl sulphoxide and related analogs.

In some embodiments, the, the agents/glucans administered as part of themethods of this invention are formulated for use as eye drops whereinthe active ingredient(s) is dissolved or suspended in a suitableexcipient(s), for example, an aqueous solvent for active ingredient(s)that comprise one or more charges at pH values near neutrality, e.g.,about pH 6-8. In some embodiments, the active ingredient(s) is presentin such formulations in a concentration of about 0.5-20% w/w, typicallyabout 1-10% w/w, often about 2-5% w/w.

In some embodiments, the agents/glucans administered as part of themethods of this invention are formulated for topical administration inthe mouth, and may include lozenges comprising the active ingredient ina flavored basis, which may comprise sucrose and acacia or tragacanth;pastilles comprising the active ingredient(s) in an inert basis such asgelatin and glycerin, or sucrose and acacia, or others; or mouthwashescomprising the active ingredient in a suitable liquid excipient(s), orothers as will be appreciated by one skilled in the art.

Formulations for rectal administration may be presented as a suppositorywith a suitable base comprising for example cocoa butter or asalicylate.

Formulations suitable for intrapulmonary or nasal administration have aparticle size for example in the range of 0.01 to 500 microns (includingaverage particle sizes in a range between 0.01 and 500 microns in 0.1micron or other increments, e.g., 0.05, 0.1, 0.5, 1, 1.5, 2.0, 2.5, 3.0,3.5, 4.0, 4.5, 5.0, 6, 7, 8, 9, 10, 20, 25, 30, 35, 50, 75, 100, etc.microns), which is administered by rapid inhalation through the nasalpassage or by inhalation through the mouth so as to reach the alveolarsacs. Suitable micronized formulations include aqueous or oily solutionsor suspensions of the active ingredient(s). Formulations suitable foraerosol, dry powder or tablet administration may be prepared accordingto conventional methods and may be delivered with other therapeuticagents such as compounds heretofore used in the treatment or prophylaxisof viral or other infections as described herein. Such formulation maybe administered, e.g., orally, parenterally (i.v. i.m., s.c), topicallyor by a buccal route.

Formulations suitable for vaginal administration may be presented aspessaries, tampons, creams, gels, pastes, foams or spray formulationscontaining in addition to the active ingredient(s) such excipients asare known in the art to be appropriate.

Formulations suitable for parenteral administration include aqueous andnon-aqueous sterile injection solutions which may contain antioxidants,buffers, bacteriostats and solutes which render the formulation isotonicwith the blood of the intended recipient; and aqueous and nonaqueoussterile suspensions which may include suspending agents and thickeningagents.

The formulations are presented in unit-dose or multi-dose containers,for example sealed ampules and vials, and may be stored in afreeze-dried (lyophilized) condition requiring only the addition of thesterile liquid excipient, for example water for injection, immediatelyprior to use. Extemporaneous injection solutions and suspensions areprepared from sterile powders, granules and tablets of the kindpreviously described. Unit dosage formulations are those containing adaily dose or unit daily sub-dose, as recited herein, or an appropriatefraction thereof, of the active ingredient(s).

It should be understood that in addition to the ingredients particularlymentioned above the formulations of this invention may include otheragents or excipients conventional in the art having regard to the typeof formulation in question, for example those suitable for oraladministration may include flavoring agents.

It will be apparent to those skilled in the art that variousmodifications and variations can be made in the compositions, use andpreparations of the present invention without departing from the spiritor scope of the invention.

For administration to mammals, and particularly humans, it is expectedthat in the case of medications, the physician or other qualified healthcare provider may determine the actual dosage and duration of treatment,which will be most suitable for an individual and can vary with the age,weight and response of the particular individual. It will be appreciatedthat in the case of nonprescription (e.g., “over-the-counter”)medications, foods, food products, food supplements, cosmetic andpersonal care compositions, the amount may be determined at thediscretion of the user, optionally with guidance from the labeling orfrom an appropriate health care provider or other advisor.

EXAMPLES Materials and Methods Preparation of IgG-Depleted Serum

Protein G-sepharose beads of untreated sepharose beads (control) werewashed three times with PBS. Serum was diluted 2-fold in PBS and addedto beads. Beads were incubated at room temperature on an end-to-endmixer for 30 min. beads were removed by centrifugation.

Serum from 10 unimmunized normal healthy donors was pooled

SRBC Assay

SRBC (Accurate chemical and scientific corp.) were washed with gelatinveronal buffer (Sigma) and opsonized with rabbit anti-SRBC antibodies(Accurate chemical and scientific corp.) for 30 minutes at roomtemperature. IgG-depleted serum (from protein G-sepharose beads) orIgG-containing serum (from untreated sepharose beads) were added to SRBCand incubated at 37° C. for one hour. Water was added as a positivecontrol (complete lysis), and buffer was added as a negative control (nolysis). Lysis was detected by direct microscopic visualization (FIG. 1Ca) or by O.D. 414 nm, which detects the heme that is secreted from thelysing SRBC).

Preparation of β-1,6-Glucan-Coated Beads and FACS Analysis (Phagocytosisand ROS Production)

These methods were performed as described in. Rubin-Bejerano, L, et al,Phagocytosis by human neutrophils is stimulated by a unique fungal cellwall component. Cell Host Microbe, 2007. 2(1): p. 55-67.

Cell Culture

SK-BR-3 cells (ATCC) were cultured in McCoy's 5A Medium (Gibco)supplemented with 10% FBS.

Conjugation

Herceptin (Genentech, Inc.) or IgG1 isotype control (Sigma) wereconjugated to β-1,6-glucan following oxidation with sodium metaperiodate (Pierce).

Neutrophils

Fresh human blood and serum were provided by Research Blood Components(Brighton, Mass.). Neutrophils were isolated from fresh human blood inaccordance with a protocol approved by the MIT Committee on Use ofHumans as Experimental Subjects by using Histopaque 1077 and Histopaque1119 (Sigma).

Opsonization

Breast cancer cells were opsonized in 40% serum in Phosphate-BufferedSaline without calcium chloride and without magnesium chloride (PBS)(Gibco) for 15 minutes at 37° C. Cells were then washed three times withcold PBS supplemented with 0.04 mg/ml of the protease inhibitor AEBSF(Sigma).

Antibody Binding and C3 Deposition

Fluorescence Activated Cell Sorting (FACS) analysis was used to detectHerceptin binding to breast cancer cells (by using anti-human IgG1antibodies, Sigma) and C3 deposition (by using anti-human C3 antibodies,Accurate Chem.).

Cytotoxicity

Cytotoxicity to breast cancer cells following incubation withβ-1,6-glucan-conjugated or unconjugated Herceptin and serum wasdetermined by CytoTox 96® Non-Radioactive Cytotoxicity Assay (Promega),which detects lactate dehydrogenase released from lysed cells. Forneutrophil-dependent killing, above cells were cultured with neutrophilsat 37° C. after which cytotoxicity was measured.

Example 1 β-1,6-Glucan Stimulated Complement Activation is DependentUpon Antibodies

In order to test whether antibodies are involved in complementactivation by β-1,6-glucan, serum used in complement activation assayswas depleted of antibodies by using protein G sepharose beads. ThisIgG-depleted serum was used to optimize β-1,6-glucan-coated beads. Thebeads were then tested in a phagocytosis assay conducted with humanneutrophils. Phagocytosis and ROS production were reduced whenβ-1,6-glucan-coated beads were opsonized with IgG-depleted serum (FIGS.1A, 1B, and 1C), suggesting that the Classical pathway plays a majorrole in complement activation by β-1,6-glucan.

The IgG-depleted serum contained functional complement factors. In asheep red blood cells (SRBC) assay, SRBC opsonized with anti-SRBCantibody were lysed when IgG-depleted serum was utilized (FIGS. 1D and1E).

Example 2 Antibodies to β-1,6-Glucan are Prevalent in Normal Adults

Pooled serum from 10 human donors, had high levels of IgG antibodiesbinding β-1,6-glucan, in contrast to low levels of IgG bindingβ-1,3-glucan. To test how prevalent is the anti-β-b 1,6-glucanantibodies in different donors, we collected sera from 12 individuals.The sera were used to opsonize β-1,6-glucan- or β-1,3-glucan-coatedbeads, and the beads were incubated with human neutrophils. Eleven ofthe twelve sera had the high response that the pool did, mediatingefficient engulfment and ROS production (FIG. 2A, a representative serumof the high responders). One serum mediated a less efficient engulfmentand ROS production (FIG. 2B, low responder).

Example 3 β-1,6-Glucan Mediates Efficient Phagocytosis and Production ofReactive Oxygen Species by Neutrophils

In order to assess which isotype of IgG was mediating the β-1,6-glucanrecognition, antibodies specific for IgG1, IgG2, IgG3, or IgG4 were usedto determine the IgG isotype usage in donor serum with a high or lowresponse to β-1,6-glucan, following exposure to opsonized13-1,6-glucan-coated beads. The high responder produced more IgG1, IgG2,and IgG3, but not IgG4, as compared to the low responder (FIGS. 3A-3F).Specifically, the level of IgG2 was dramatically higher in the highresponder, probably because polysaccharides tend to induce production ofthe IgG2 isotype.

The different isotypes differ in their complement activation properties.IgG3 has the highest complement activation properties, then followsIgG1, IgG2 is next, and IgG4 cannot activate complement. IgG2 is a pooractivator of the Classical pathway, but it can activate complementthrough the alternative pathway, and is a good substrate forphagocytosis by neutrophils.

Example 4 β-4,6-Glucan Conjugated to the Herceptin Monoclonal AntibodyMediates Recruitment of Complement and Neutrophils to Breast CancerCells

In order to assess whether conjugated β-1,6-glucan linked to targetingmoieties can mediate recruitment of complement and neutrophils to lyseand kill target cells bound by the targeting moieties, a 1,6-glucanlinked to a Herceptin monoclonal antibody (mAb) was tested in a systemof breast cancer cells (SK-BR-3). The Herceptin mAb is directed againstthe Her-2/neu protein overexpressed on SK-BR-3 cells. Conjugation ofJβ-1,6-glucan to Herceptin did not affect its binding to breast cancercells (FIG. 4A). Furthermore, the conjugate mediated high C3 deposition(FIG. 4B), suggesting that β-1,6-glucan remained functional.Non-specific isotype control antibodies conjugated to β-1,6-glucan didnot bind breast cancer cells (FIG. 4C), and therefore, did not mediatecomplement activation and C3 deposition on these cells (FIG. 4D). Mixingβ-1,6-glucan with the mAb without chemical conjugation did not mediateC3 deposition on these breast cancer cells (FIG. 4E, compare green toblue). Therefore, it was concluded that the conjugation of β-1,6-glucanto the targeting moiety was required for directing of complement andconsequently neutrophils to target cells. C3 deposition was detected onbreast cancer cells treated with Herceptin conjugated to β-1,6-glucanbut not β-1,3-glucan (FIG. 4F, compare blue to green), suggesting thatindeed β-1,6-glucan was more efficient than β-1,3-glucan in attractingcomplement.

Example 4 β-1,6-Glucan Conjugated to the Herceptin Monoclonal AntibodyMediates Killing of Breast Cancer Cells by Complement and Neutrophils

Deposition of the complement protein C3 on breast cancer cells led tolysis of these cancer cells. The Herceptin-1,6-glucan conjugate showed adose-dependent cytotoxic effect on the breast cancer cells, whereas theunconjugated Herceptin lacked any effect (FIG. 5 A). Furthermore, theHerceptin-β-1,6-glucan conjugate showed an increased neutrophil killingof the cancer cells (FIG. 5 B).

VIII. EQUIVALENTS AND SCOPE

It will be understood by those skilled in the art that various changesin form and details may be made therein without departing from thespirit and scope of the invention as set forth in the appended claims.Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed in the scope of the claims.

In the claims articles such as “a,” “an” and “the” mean one or more thanone unless indicated to the contrary or otherwise evident from thecontext. Claims or descriptions that include “or” or “and/or” betweenmembers of a group are considered satisfied if one, more than one, orall of the group members are present in, employed in, or otherwiserelevant to a given product or process unless indicated to the contraryor otherwise evident from the context. The invention includesembodiments in which exactly one member of the group is present in,employed in, or otherwise relevant to a given product or process. Theinvention also includes embodiments in which more than one, or all ofthe group members are present in, employed in, or otherwise relevant toa given product or process. Furthermore, it is to be understood that theinvention provides, in various embodiments, all variations,combinations, and permutations in which one or more limitations,elements, clauses, descriptive terms, etc., from one or more of thelisted claims is introduced into another claim dependent on the samebase claim unless otherwise indicated or unless it would be evident toone of ordinary skill in the art that a contradiction or inconsistencywould arise. Where elements are presented as lists, e.g. in Markushgroup format or the like, it is to be understood that each subgroup ofthe elements is also disclosed, and any element(s) can be removed fromthe group. It should it be understood that, in general, where theinvention, or aspects of the invention, is/are referred to as comprisingparticular elements, features, etc., certain embodiments of theinvention or aspects of the invention consist, or consist essentiallyof, such elements, features, etc. For purposes of simplicity thoseembodiments have not in every case been specifically set forth in haecverba herein. Certain claims are presented in dependent form for thesake of convenience, but Applicant reserves the right to rewrite anydependent claim in independent format to include the elements orlimitations of the independent claim and any other claim(s) on whichsuch claim depends, and such rewritten claim is to be consideredequivalent in all respects to the dependent claim in whatever form it isin (either amended or unamended) prior to being rewritten in independentformat.

1.-123. (canceled)
 124. A method of treating a subject with a vaccine,adjuvant or composition comprising β-1,6-glucan, the method comprisingadministering a vaccine, adjuvant or composition comprising β-1,6-glucanto a subject, wherein the subject has previously been determined topossess a higher titer of IgG1 or IgG2 or IgG3, or a combinationthereof, versus a titer of IgG4 to β-1,6-glucan.
 125. The method ofclaim 124, wherein the subject has previously been determined to possessa higher titer of IgG2 versus IgG4 to β-1,6-glucan.
 126. The method ofclaim 124, wherein the vaccine, adjuvant or composition comprisesβ-1,6-glucan linked to an antibody.
 127. The method of claim 126,wherein the antibody is a monoclonal antibody.
 128. The method of claim126, wherein the antibody is a human antibody.
 129. The method of claim126, wherein the antibody is a humanized antibody.
 130. The method ofclaim 126, wherein the antibody is an antibody fragment.
 131. The methodof claim 130, wherein the antibody fragment is a Fab fragment, Fab′fragment, (Fab′)₂ fragment, Fv fragment, a single chain antibody or apeptide coding for a single complementarity-determining region.
 132. Themethod of claim 126, wherein the antibody is selected from the groupconsisting of Alemtuzumab, Bevacizumab, Cetuximab, Gemtuzumab,Ibritumomab, Panitumumab, Rituximab, Tositumomab, Trastuzumab andPalivizumab.
 133. The method of claim 126, wherein the antibody targetsa neoplastic or preneoplastic cell.
 134. The method of claim 133,wherein the vaccine, adjuvant or composition comprising β-1,6-glucanpromotes a host anticancer response.
 135. The method of claim 133,wherein the vaccine, adjuvant or composition comprising β-1,6-glucanpromotes tumor cell lysis.
 136. The method of claim 133, wherein thevaccine, adjuvant or composition comprising β-1,6-glucan enhances a hostantitumor response.
 137. The method of claim 126, wherein the antibodytargets an antigen expressed specifically on cancer cells.
 138. Themethod of claim 137, wherein the vaccine, adjuvant or compositioncomprising β-1,6-glucan enhances complement-mediated lysis of the cancercells.
 139. A method of treating a subject with a vaccine, adjuvant orcomposition comprising β-1,6-glucan, the method comprising:administering an agent to a subject which biases antibody production toyield relatively greater amounts of IgG1 or IgG2 or IgG3, or acombination thereof, versus IgG4, wherein the subject has previouslybeen determined to possess a lower titer of IgG1 or IgG2 or IgG3, or acombination thereof, versus IgG4 to β-1,6-glucan; and administering avaccine, adjuvant or composition comprising β-1,6-glucan to the subjectwhen the subject possesses a higher titer of IgG1 or IgG2 or IgG3, or acombination thereof, versus a titer of IgG4 to β-1,6-glucan.
 140. Themethod of claim 139, wherein the agent is selected from the groupconsisting of cytokines, chemokines, complement components, immunesystem accessory and adhesion molecules and receptors of any of these.141. The method of claim 140 wherein the agent is selected from thegroup consisting of interleukin 2, interleukin 12, interferon-gamma andcombinations thereof.
 142. The method of claim 139, wherein the agent isadministered based on a determination that the subject possesses a lowertiter of IgG2 versus IgG4 to β-1,6-glucan.
 143. The method of claim 126,wherein the composition is a pharmaceutical composition.
 144. The methodof claim 124, wherein the vaccine, adjuvant or composition comprisesβ-1,6-glucan linked to an aptamer.
 145. The method of claim 124, whereinthe vaccine, adjuvant or composition comprises β-β-1,6-glucan linked toa peptide.
 146. A method of treating a subject with a vaccine, adjuvantor composition comprising β-1,6-glucan, the method comprisingidentifying a subject that possesses a higher titer of IgG1 or IgG2 orIgG3, or a combination thereof, versus a titer of IgG4 to β-1,6-glucanand administering a vaccine, adjuvant or composition comprisingβ-1,6-glucan to the subject.
 147. The method of claim 146, wherein thesubject possesses a higher titer of IgG2 versus IgG4 to β-1,6-glucan.148. The method of claim 147, wherein the vaccine, adjuvant orcomposition comprises β-1,6-glucan linked to an antibody.
 149. Themethod of claim 148, wherein the composition is a pharmaceuticalcomposition.
 150. A method of treating a subject with a vaccine,adjuvant or composition comprising β-1,6-glucan, the method comprisingadministering a vaccine, adjuvant or composition comprising β-1,6-glucanto a subject, wherein the subject has previously been determined topossess a level of IgG2 titer to β-1,6-glucan above a threshold. 151.The method of claim 150, wherein the vaccine, adjuvant or compositioncomprises β-1,6-glucan linked to an antibody.
 152. The method of claim151, wherein the composition is a pharmaceutical composition.
 153. Amethod of treating a subject with a vaccine, adjuvant or compositioncomprising β-1,6-glucan, the method comprising identifying a subjectthat possesses a level of IgG2 titer to β-1,6-glucan above a thresholdand administering a vaccine, adjuvant or composition comprisingβ-1,6-glucan to the subject.
 154. The method of claim 153, wherein thevaccine, adjuvant or composition comprises β-1,6-glucan linked to anantibody.
 155. The method of claim 154, wherein the composition is apharmaceutical composition.